Yeast-Based Therapeutic for Chronic Hepatitis C Infection

ABSTRACT

Disclosed are compositions, including vaccines, and methods for vaccinating an animal against hepatitis C virus (HCV) and for treating or preventing hepatitis C viral infection in an animal. The invention includes a variety of novel HCV fusion proteins that can be used directly as a vaccine or in conjunction with a yeast-based vaccine vehicle to elicit an immune response against HCV in an animal. The invention also includes the use of the HCV fusion gene and protein described herein in any diagnostic or therapeutic protocol for the detection and/or treatment or prevention of HCV infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. patent applicationSer. No. 11/618,834, filed Dec. 31, 2006, which will issue as U.S. Pat.No. 7,632,511, which claims the benefit of priority under 35 U.S.C. §120and is a continuation of PCT Application Serial No. PCT/US2005/037499,filed Oct. 18, 2005, now expired, which designates the United States,and which claims the benefit of priority under 35 U.S.C. §119(e) fromU.S. Provisional Application Ser. No. 60/620,158, filed on Oct. 18,2004, now expired. The entire disclosure of each of PCT Application No.PCT/US2005/037499 and of U.S. patent application Ser. No. 11/618,834 isincorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “3923-12-2-1_ST25”, has asize in bytes of 101 KB, and was recorded on 4 Dec. 2009. Theinformation contained in the text file is incorporated herein byreference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

This invention generally relates to compositions and methods forvaccinating an animal against hepatitis C virus (HCV) and for treatingor preventing hepatitis C viral infection in an animal.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a major causative agent of acute and chronichepatitis worldwide. It is estimated that there are 200 millionchronically HCV-infected individuals worldwide, 4 million of whom residein the United States. The foremost source of infection is throughparenteral routes including blood transfusions or IV drug use. Despitethe high degree of safety associated with current blood bankingprocedures, the rate of infection continues to increase, presumably dueto IV drug use and other forms of exposure.

According to data from the Third National Health and NutritionExamination Survey (NHANES III), approximately 70% of the patients withHCV infections in the United States will become chronically infected. Asignificant proportion of chronically infected individuals will suffer aserious sequelae of chronic HCV infection including progression tocirrhosis, hepatic decompensation, liver transplant, hepatocellularcarcinoma, and death. Retrospective long term follow-up studies onpatients chronically infected with HCV estimate the proportion who willprogress to cirrhosis at approximately 20% to 50% with follow-up timesranging from 10 to 29 years (1-4). Prospective long term follow-upstudies on patients chronically infected with HCV after post-transfusionexposure estimate the proportion who will progress to cirrhosis atapproximately 10% to 15% with relatively short follow-up times rangingfrom 8 to 16 years (5-8). Of those patients who develop cirrhosissecondary to viral infection it is predicted that approximately 1% to3%, will develop hepatocellular carcinoma annually with an approximateannual mortality rate of 2% to 6% (9-10). An epidemiologic modelutilizing NHANES III seroprevalance data and age-specific incidencerates estimates a peak in U.S. population risk for progression tocirrhosis and related complications by 2015, foretelling of a worseningunmet medical need in the near future (11). Interruption of the chronicviral infection using interferon based regimens has been shown inseveral large series to favorably alter the rates of progression tocirrhosis, hepatocellular, and death (12-14). However, sustainedvirologic response rates for the treatment of genotype 1 chronichepatitis C, the predominant genotype found in the US, are onlyapproximately 50% with pegylated interferon-α regimens containingribavirin. Additionally, interferon plus ribavirin based regimens alsohave significant safety problems including depression, suicidalideation, flu-like symptoms, and neutropenia. Treatment options arecurrently limited for partial responders, relapsers, and non-respondersto interferon based therapy.

HCV is a member of the Flaviviridae family of enveloped, positive-senseRNA viruses. It has a genome of approximately 9600 nucleotides that istranslated upon cell entry into a polyprotein of roughly 3000 aminoacids. Three structural and seven non-structural proteins are generatedco- and post-translationally by cellular and HCV-derived proteases(Table 1). While the roles of some of the viral proteins have yet to beclearly defined, a number of them, such as the HCV structural Coreprotein, the E1 and E2 surface glycoproteins, the non-structural NS2 andNS3 proteases, and the NS5B RNA-dependent RNA polymerase are known toperform essential functions in the HCV life cycle. Based on geneticheterogeneity of the viral genomes isolated so far, HCV has 6 majorgenotypes and more than 100 subtypes.

Genotypes 1a, 1b and 2 are found predominantly in North America andEurope, while in South America, HCV genotypes 1a, 1b, and 3 areprevalent. Genotypes 4, 5 and 6 are observed throughout the rest of theworld (19). Despite the geographic predominance of certain HCVgenotypes, most genotypes have been identified all over the world due toincreased population movement. The different HCV genotypes vary in termsof their response to the currently recommended interferon/ribavirintherapy. In particular, ˜50% of patients infected with HCV genotype 1remain refractory to the current treatment regimen (19). Further,response rates to interferon alpha among African-American patients arelower than those of Caucasian descent. These data suggest the need foralternative treatments that ideally augment the individual'spre-existing cellular immune response.

TABLE 1 HCV genes and gene products % homology between HCV Gene Functiongenotypes 1a and 1b Core Nucleocapsid core protein 98.4 E1 Envelopeglycoprotein 81.8 E2 Envelope glycoprotein 79.9 P7 Ion channel 81.0 NS2metalloprotease 80.1 NS3 protease/helicase 92.1 NS4a NS3 proteaseco-factor 91.1 NS4b Unknown 82.4 NS5a Unknown 77.7 NS5b RNA-dependentRNA polymerase 87.5 The HCV protein sequences were obtained from theNational Center for Biotechnology Information under Accession No.AF011753 (gi: 2327074). The Align program from the GenestreamBioinformatics website (Institut de Génétique Humaine, 141 rue de laCardonille, Montpellier France) was used to compare the amino acidsequences of the HCV proteins derived from strain 1a and 1b.

Numerous studies suggest that viral replication, the level of viremiaand progression to the chronic state in HCV-infected individuals areinfluenced directly and indirectly by HCV-specific cellular immunitymediated by CD4⁺ helper (T_(H)) and CD8⁺ cytotoxic T lymphocytes (CTLs),and directed against both structural and non-structural viral proteinsincluding Core and NS3 (15). The lack of effective immunity in personswith chronic HCV infection is further implied by the occurrence ofsuperinfection with other genotypes of HCV. As the robustness andbreadth of cellular immune responses have been suggested to influencethe natural course of HCV infection, the development ofimmunotherapeutic products that stimulate T cell immune responses invirally exposed individuals is of major importance.

Studies of humans and chimpanzees have revealed that HCV can replicatefor weeks before the onset of CD4⁺ and CD8⁺ T cell responses in bloodand liver. Moreover, there may be a delay in the acquisition of functionby CD8⁺ (and perhaps CD4⁺) T cells even after their expansion in blood(15). The appearance of functional CD8⁺ T cells is kineticallyassociated with control of viremia and, at least in some cases, with anelevation in serum transaminases, suggesting that liver damage duringacute hepatitis C is immunopathological. At highest risk of persistentHCV infection are those individuals who fail to generate a detectablevirus-specific T lymphocyte response in the blood, liver, or both.Perhaps most importantly, generation of a cellular immune response doesnot necessarily ensure that the infection will be permanentlycontrolled. CD4⁺ and CD8⁺ T cell responses must be sustained for weeksor months beyond the point of apparent control of virus replication toprevent relapse and establishment of a persistent infection.

CD4⁺ T cells play an essential role in anti-HCV immunity by providinghelp for activating and sustaining CD8⁺ T cell responses. ProtectiveCD4⁺ T cells appear to predominantly recognize epitopes in Core, NS3,NS4 and NS5 proteins although responses against the other HCV geneproducts have also been reported (20-21). In addition to the help thatCD4⁺ T cells provide to CD8⁺ T cells, it also appears critical that theyproduce gamma interferon and other pro-inflammatory T_(H)1-, as opposedto, T_(H)2-type cytokines Equally important for control of chronicinfection is the establishment of HCV-specific memory CD4⁺ T cells (20 &22).

The finding that CD4⁺ and CD8⁺ T cell responses are common toself-limited HCV infections suggests that they cooperate to bring aboutcontrol of viremia. Memory CD4⁺ and CD8⁺ T cells primed during acuteresolving hepatitis C infection provide long-term protection from viruspersistence in chimpanzees and probably humans. Throughantibody-mediated depletion of each memory T cell subset, the chimpanzeemodel has provided direct proof of the importance of CD8⁺ T cells in thecontrol of acute hepatitis C and their dependence on CD4⁺ T cell help(24). In contrast to CD4⁺ T cells, both acute and memory CD8⁺ T cellsappear to recognize all of the HCV proteins equally and, as with CD4⁺ Tcells, it may be critical that they be capable of producingpro-inflammatory cytokines including gamma interferon (15).

The transition from acute to chronic HCV infection is associated withsubstantial loss of HCV-specific CD4⁺ T cells that do not appear torecover during the life of the host. CD8⁺ T cell activity is alsoimpaired, as it is insufficient for resolution of infection.

A number of experimental approaches to immunotherapy in general havebeen investigated, including the use of DNA-, recombinant viral-, andautologous dendritic cell-based vaccine strategies. DNA vaccines aregood at priming immune responses in humans but are poor at boosting. Incontrast, recombinant viruses are good at boosting but suffer from thelimitation of vector neutralization. Finally, dendritic cell-basedvaccines are patient-specific and labor intensive. Therefore, thereremains a need in the art for an effective immunotherapeutic approachagainst HCV.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a vaccine comprising:(a) a yeast vehicle; and (b) an HCV fusion protein, wherein the yeastvehicle recombinantly expresses the fusion protein. The HCV fusionprotein can be chosen from any of the HCV fusion proteins describedbelow, with an HCV fusion protein comprising at least a portion of anHCV NS3 protease linked to at least a portion of an HCV Core sequence,being particularly preferred.

Accordingly, in one aspect, the HCV fusion protein comprises at least aportion of an HCV NS3 protease linked to at least a portion of an HCVCore sequence. Preferably, the HCV NS3 protease lacks the catalyticdomain of a natural HCV NS3 protease. In one aspect, the HCV NS3protease consists essentially of the 262 amino acids of HCV NS3following the initial N-terminal 88 amino acids of the full-length NS3protein (positions 1115 to 1376 with respect to SEQ ID NO:20). In oneaspect, the hydrophobic C-terminal sequence of the HCV Core istruncated. In one aspect, the HCV Core sequence consists essentially ofamino acid positions 2 through 140 of the full-length HCV Core sequence(positions 2 to 140, with respect to SEQ ID NO:20). In another aspect,the HCV Core sequence has been appended to include two amino acids,glutamate and aspartate. In another aspect, the HCV Core sequence hasbeen appended to include the amino acid sequence of G-G-G-H-H-H-H-H-H(SEQ ID NO:10). In one aspect, the HCV NS3 protease is linked at itsN-terminus to the amino acid sequence represented by SEQ ID NO:9(MADEAP). In yet another aspect, the fusion protein consists essentiallyof SEQ ID NO:2.

In another aspect, the fusion protein comprises a full-length,inactivated HCV NS3 protein. In one aspect, the HCV NS3 proteincomprises a mutation at residue 1165 of the HCV polyprotein sequence,with respect to SEQ ID NO:20, that results in inactivation of theproteolytic activity of the protein. In one aspect, the HCV fusionprotein comprises an HCV NS3 protease sequence linked to a heterologousamino acid sequence, wherein the HCV NS3 protease sequence consistsessentially of positions 1027 to 1657 of SEQ ID NO:20, wherein theserine residue corresponding to position 1165 of SEQ ID NO:20 has beenmutated to inactivate the proteolytic activity of the protein. In oneaspect, the serine corresponding to position 1165 of SEQ ID NO:20 hasbeen substituted with an alanine residue. In another aspect, the HCV NS3protease is linked at its N-terminus to the amino acid sequencerepresented by SEQ ID NO:9 (MADEAP). In yet another aspect, the fusionprotein consists essentially of SEQ ID NO:4.

In yet another aspect, the fusion protein comprises a truncated HCV E1protein fused to a truncated HCV E2 protein. In one aspect, thetruncated HCV E1 protein consists essentially of amino acids 1 to 156 ofHCV E1 (positions 192 to 347, with respect to SEQ ID NO:20). In yetanother aspect, the truncated HCV E2 protein consists essentially ofamino acids 1 to 334 of HCV E2 (positions 384 to 717, with respect toSEQ ID NO:20). In yet another aspect, the truncated HCV E1 protein islinked at its N-terminus to the amino acid sequence represented by SEQID NO:9 (MADEAP). In another aspect, the fusion protein consistsessentially of SEQ ID NO:6.

In another aspect, the fusion protein comprises a transmembranedomain-deleted HCV NS4b protein. In one aspect, the transmembranedomain-deleted HCV NS4b protein consists essentially of amino acids 1 to69 of HCV NS4b (positions 1712 to 1780, with respect to SEQ ID NO:20)linked to amino acids 177 to 261 of HCV NS4b (positions 1888 to 1972,with respect to SEQ ID NO:20). In yet another aspect, the transmembranedomain-deleted HCV NS4b protein is linked at its N-terminus to the aminoacid sequence represented by SEQ ID NO:9 (MADEAP). In another aspect,the fusion protein consists essentially of SEQ ID NO:8.

In yet another aspect, the fusion protein comprises a full-length HCVCore protein fused to a full-length HCV E1 protein fused to afull-length HCV E2 protein. In one aspect, the full-length HCV Coreprotein is linked at its N-terminus to the amino acid sequencerepresented by SEQ ID NO:9 (MADEAP). In another aspect, the fusionprotein consists essentially of SEQ ID NO:12.

In another aspect, the fusion protein comprises a truncated HCV Coreprotein fused to an HCV E1 protein with deleted transmembrane domain andan HCVE2 protein with deleted transmembrane domain. In one aspect, thetruncated HCV Core protein consists essentially of positions 2 to 140 ofHCV Core protein (positions 2 to 140, with respect to SEQ ID NO:20). Inanother aspect, the HCV E1 protein with deleted transmembrane domainconsists essentially of positions 1 to 156 of HCV E1 protein (positions192 to 347, with respect to SEQ ID NO:20). In yet another aspect, thetruncated HCV E2 protein consists essentially of positions 1 to 334 ofHCV E2 protein (positions 384 to 717, with respect to SEQ ID NO:20). Inyet another aspect, the truncated HCV Core protein is linked at itsN-terminus to the amino acid sequence represented by SEQ ID NO:9(MADEAP). In another aspect, the fusion protein consists essentially ofSEQ ID NO:14.

In another aspect, the fusion protein comprises HCV NS3 fused to HCVNS4a fused to HCV NS4b, wherein the HCV NS3 protease is inactivated andthe HCV NS4b lacks a transmembrane domain. In one aspect, the HCV NS3protein consists essentially of positions 1 to 631 of HCV HS3 (positions1027 to 1657, with respect to SEQ ID NO:20), wherein the serine atposition 1165 with respect to SEQ ID NO:20 has been substituted withalanine, to inactivate the protease. In one aspect, the HCV NS4a proteinconsists essentially of positions 1 to 54 of the HCV NS4a protein(positions 635 to 691, with respect to SEQ ID NO:20). In yet anotheraspect, the HCV NS4b protein consists essentially of positions 1 to 69of HCV NS4b (positions 1712 to 1780, with respect to SEQ ID NO:20) fusedto positions 177 to 261 of HCV NS4b (positions 1888 to 1972, withrespect to SEQ ID NO:20). In another aspect, the HCV NS3 protein islinked at its N-terminus to the amino acid sequence represented by SEQID NO:9 (MADEAP). In yet another aspect, the fusion protein consistsessentially of SEQ ID NO:16.

In yet another aspect, the fusion protein comprises an HCV NS5a proteinfused to an HCV NS5b protein, wherein the NS5b protein contains aninactivating deletion of NS5b C-terminus. In one aspect, the HCV NS5aprotein consists essentially of 1 to 448 of HCV NS5a (positions 1973 to2420, with respect to SEQ ID NO:20). In one aspect, the HCV NS5b proteinconsists essentially of positions 1 to 539 of HCV NS5b (positions 2421to 2959, with respect to SEQ ID NO:20). In yet another aspect, the HCVNS5a protein is linked at its N-terminus to the amino acid sequencerepresented by SEQ ID NO:9 (MADEAP). In yet another aspect, the fusionprotein consists essentially of SEQ ID NO:18.

In one embodiment, the expression of the fusion protein is under thecontrol of an inducible promoter, such as CUP1.

Another embodiment of the present invention relates to an isolated HCVfusion protein, wherein the HCV protein is any of the above-describedproteins and particularly, is chosen from SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 and SEQID NO:18.

Another embodiment of the present invention relates to an isolatednucleic acid molecule comprising a nucleic acid sequence encoding any ofthe above-described fusion proteins. In one embodiment, the expressionof the fusion protein is under the control of an inducible promoter,such as CUP1.

Another embodiment of the present invention relates to a recombinantnucleic acid molecule comprising any of such isolated nucleic acidmolecules. In one embodiment, the recombinant nucleic acid molecule is aviral vector.

Yet another embodiment of the invention relates to a recombinant cellthat has been transfected with any of the recombinant nucleic acidmolecules described herein. Such a cell can include, but is not limitedto, a tumor cell or a yeast cell.

Another embodiment of the present invention relates to a vaccinecomprising: (a) an HCV fusion protein as described above; and (b) apharmaceutically acceptable carrier.

Yet another embodiment of the present invention relates to a vaccinecomprising: (a) a dendritic cell; and (b) an HCV fusion protein asdescribed above. Such a vaccine can further comprise a yeast vehicle,wherein the dendritic cell also contains the yeast vehicle.

Yet another embodiment of the present invention relates to a vaccinecomprising an isolated nucleic acid molecule encoding an HCV fusionprotein as described above.

Any of the above-described vaccines of the invention that include anisolated HCV fusion protein of the invention can also include at leastone biological response modifier. Such biological response modifiers caninclude, but are not limited to: a cytokine, a hormone, a lipidicderivative, and a small molecule drug. Such biological responsemodifiers can include, but are not limited to: anti-CTLA-4, anti-CD137,anti-CD28, anti-CD40, alemtuzumab, denileukin diftitox, anti-CD4,anti-CD25, anti-PD1, anti-PD-L1, anti-PD-L2, FOXP3-blocking agents,Flt-3 ligand, imiquimod, granulocyte-macrophage colony-stimulatingfactor (GM-CSF), sargramostim, Toll-like receptor (TLR)-7 agonists, andTLR-9 agonists.

Another embodiment of the present invention, relates to a method toprotect an animal against hepatitis C virus (HCV) infection, comprisingadministering to an animal that has been infected with HCV or is at riskof being infected with HCV, any of the vaccines of the present inventionas described herein, wherein administration of the vaccine to the animalreduces or prevents HCV infection or at least one symptom resulting fromHCV infection in the animal.

Yet another embodiment of the present invention relates to a method toelicit an antigen-specific, cell-mediated immune response against an HCVantigen, comprising administering to an animal any of the vaccines ofthe present invention as described herein.

Another embodiment of the present invention relates to a method toelicit an antigen-specific, cell-mediated immune response against an HCVantigen in a population of individuals who have been infected with HCV,comprising administering to said population of individuals any of theabove-described vaccines.

Yet another embodiment of the present invention relates to a method toimmunize against HCV a population of individuals that is at risk ofbecoming infected with HCV, comprising administering to said populationof individuals a vaccine according to any of the above-describedvaccines.

In any of the above methods, the vaccine can be administered as abooster to a vaccine comprising a viral vector encoding an HCV antigen.In either of the above-methods, the vaccine can be administered to primethe immune system prior to boosting with a different HCV vaccine.

Another embodiment of the present invention relates to the use of any ofthe above-described vaccines in a formulation for protecting an animalagainst HCV infection.

Yet another embodiment of the present invention relates to the use ofany of the above-described vaccines in a formulation for eliciting anantigen-specific, cell-mediated immune response against an HCV antigen.

Another embodiment of the present invention relates to the use any ofthe above-described vaccines in a formulation for treating or preventinga disease or condition.

Yet another embodiment of the present invention relates to the use ofany of the above-described vaccines in a formulation for immunizing apopulation of individuals at risk for becoming infected with HCV.

Another embodiment of the present invention relates to the use any ofthe above-described vaccines in a formulation for treating a populationof individuals that are infected with HCV.

BRIEF DESCRIPTION OF THE DRAWINGS OF THE INVENTION

FIGS. 1A and 1B are digital images of a Western blot (FIG. 1A) andCoomassie stain (FIG. 1B) showing expression of a truncated NS3-Corefusion protein and an inactivated HCV NS3 fusion protein in yeastvehicles according to the present invention.

FIG. 1C is a digital image of a Western blot showing expression of atruncated HCV E1-E2 fusion protein in a yeast vehicle according to thepresent invention.

FIG. 1D is a digital image of a Western blot showing expression of atransmembrane (TM) domain-deleted HCV NS4b fusion protein in a yeastvehicle according to the present invention.

FIG. 2 is a graph illustrating that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces NS3- andCore-specific lymphocyte proliferation.

FIGS. 3A-3C are graphs illustrating that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces NS3-specificcytotoxic effector cells.

FIGS. 4A and 4B are graphs demonstrating that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces cytotoxiceffector cells that kill tumor cells infected with recombinant vacciniavirus encoding HCV NS3 or Core.

FIG. 5 is a graph illustrating that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces secretion ofpro-inflammatory cytokines by mouse splenocytes.

FIG. 6 is a graph showing proliferating lymphocytes induced by one, twoor three weekly immunizations with a vaccine of the invention expressinga truncated NS3-Core fusion protein.

FIGS. 7A-7D are graphs showing the cytotoxic effector cell activityinduced by one, two or three weekly immunizations with a vaccine of theinvention expressing a truncated NS3-Core fusion protein.

FIGS. 8A and 8B are graphs showing pro-inflammatory yeast-specificcytokine-secreting cells induced by one, two or three weeklyimmunizations with a vaccine of the invention expressing a truncatedNS3-Core fusion protein.

FIG. 9 is a graph illustrating lymphocyte proliferation in spleen cellsderived from BALB/c mice that that were immunized and boosted with avaccine of the invention expressing a truncated NS3-Core fusion proteinunder different immunization protocols.

FIG. 10 is a graph illustrating cytotoxic effector cell activity inspleen effector cells derived from the BALB/c mice that were immunizedand boosted with a vaccine of the invention expressing a truncatedNS3-Core fusion protein under different immunization protocols.

FIGS. 11A and 11B are graphs demonstrating the durability of lymphocyteproliferative responses induced with a vaccine of the inventionexpressing a truncated NS3-Core fusion protein.

FIGS. 12A and 12B are graphs showing the durability of cytotoxiceffector cell responses induced with a vaccine of the inventionexpressing a truncated NS3-Core fusion protein.

FIGS. 13A-13D are graphs showing the durability of yeast- andNS3-specific cytokine-secreting cells induced with a vaccine of theinvention expressing a truncated NS3-Core fusion protein.

FIGS. 14A-14I are graphs illustrating cytotoxic effector cell activityinduced with a vaccine of the invention expressing different amounts ofa truncated NS3-Core fusion protein.

FIGS. 15A-15C are graphs showing pro-inflammatory cytokine secretingcells induced with a vaccine of the invention expressing differentamounts of a truncated NS3-Core fusion protein.

FIG. 16 is a graph showing that vaccines of the invention expressing atruncated NS3-Core fusion protein or an inactivated HCV NS3 proteasefusion protein induces protective immunity in BALB/c mice againstchallenge with syngeneic tumor cells expressing HCV NS3.

FIG. 17 is a graph illustrating that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces protectiveimmunity in C57BL/6 mice against challenge with syngeneic tumor cellsexpressing HCV NS3.

FIG. 18 is a graph showing lymphocyte proliferative activity in spleencells from “protected” mice.

FIG. 19 is a graph showing cytotoxic effector cell activity in spleencells from “protected” mice.

FIG. 20 is a graph illustrating that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein stimulates cytotoxiceffector cell activity in spleen cells isolated from naïve tumor-bearingmice.

FIG. 21 is a graph showing that a vaccine of the invention expressing atruncated NS3-Core fusion protein induces therapeutic immunity in BALB/cmice bearing syngeneic B cell lymphomas expressing HCV NS3.

FIGS. 22A and 22B are graphs showing that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces therapeuticimmunity in BALB/c mice bearing syngeneic B cell lymphomas expressingHCV NS3.

FIGS. 23A and 23B are graphs showing that a vaccine of the inventionexpressing a truncated NS3-Core fusion protein induces yeast-specificlymphocyte proliferation in male and female New Zealand White Rabbits.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to compositions and methods forvaccinating an animal against hepatitis C virus (HCV) and for treatingor preventing hepatitis C viral infection in an animal. The inventionincludes the use of a particular yeast-based vaccine comprising a yeastvehicle and an HCV antigen fusion protein that is selected to elicit animmune response against HCV infection in an animal. The invention alsoincludes the use of the HCV fusion gene and protein described herein inany vaccine and vaccine protocol for HCV.

Clinical evidence suggests that clearance and control of hepatitis Cvirus (HCV) infection is facilitated by cell-mediated immunity and thatenhancement of immunity in chronically-infected individuals may havetherapeutic benefits. Previous studies reported by the present inventorsand others have shown the potential for using whole, recombinant S.cerevisiae yeast as a vaccine and immunotherapy vector (e.g., see U.S.Pat. No. 5,830,463, issued Nov. 3, 1998, U.S. patent application Ser.No. 09/991,363, filed Nov. 15, 2001, each of which is incorporatedherein by reference in its entirety). The present inventors' yeast-basedimmunotherapeutic products have been shown to elicit immune responsesthat are capable of killing target cells expressing a variety of viraland cancer antigens in vivo, in a variety of animal species, and to doso in an antigen-specific, CD8⁺ CTL-mediated fashion (16-17).

The present invention is directed to an improvement on the platformtechnology related to yeast-based immunotherapeutic products asdescribed in U.S. Pat. No. 5,830,463, issued Nov. 3, 1998; U.S. patentapplication Ser. No. 09/991,363, filed Nov. 15, 2001. The presentinventors have previously shown that S. cerevisiae are avidlyphagocytosed by and directly activate dendritic cells which then presentyeast-associated proteins to CD4 and CD8 T cells in a highly efficientmanner (Stubbs et al. Nature Med. 5:625-629, 2001; and U.S. patentapplication Ser. No. 09/991,363, supra). S. cerevisiae that expressmutant Ras oncoproteins were shown to specifically eliminate establishedtumors bearing the homologous mutations in a mouse model of spontaneouslung cancer (Lu et al., Cancer Research 64:5084-5088, 2004) and thisapproach is currently being tested in a phase 1 human clinical trial inpatients with pancreatic, lung and colorectal cancer. Immunotherapeuticproducts based on this platform technology are straightforward toproduce, are not neutralized by host immune responses, can beadministered repeatedly to boost antigen-specific immune responses, anddo not require a patient-specific approach for manufacturing.

More particularly, and by way of example, the present inventors havedeveloped a yeast-based vaccine that comprises a recombinantheat-inactivated S. cerevisiae yeast expressing a novel HCV fusionprotein, which in one embodiment, contains at least a portion of bothNS3 and Core protein sequences. Other embodiments include a novelfull-length inactivated NS3 HCV protein, a novel truncated E1-E2 fusionprotein, and a novel TM domain-deleted HCV NS4b fusion protein. Otherembodiments of the invention will be apparent in view of the disclosureprovided herein.

The HCV Core protein and NS3 protease are abundantly expressed inHCV-infected cells and are essential for virus replication; thesecharacteristics combined with the high degree of sequence conservationmake them excellent targets for immunotherapy. The vaccine of thepresent invention has been shown in animals to generate both antigenspecific proliferative T cell responses as well as cytotoxic T cell(CTL) responses against virally infected cells expressing both NS3 andCore antigens and to protect animals against tumors expressing HCVantigens (see Examples and 18). Administration of the vaccine isexpected to augment the HCV-specific CD4⁺ and CD8⁺ T cell responsetargeted to the HCV NS3 and Core proteins, result in a reduction ofviral load, and ultimately lead to viral clearance in HCV-infectedindividuals.

The novel HCV fusion protein that is used as a component of theyeast-based vaccine of the present invention is produced using a novelconstruct for expression of heterologous antigens in yeast, wherein thedesired antigenic protein(s) or peptide(s) are fused at theiramino-terminal end to: (a) a specific synthetic peptide describedherein; or (b) at least a portion of an endogenous yeast protein,wherein either fusion partner provides significantly enhanced stabilityof expression of the protein in the yeast and/or a preventspost-translational modification of the proteins by the yeast cells.Also, the fusion peptides provide an epitope that can be designed to berecognized by a selection agent, such as an antibody, and do not appearto negatively impact the immune response against the vaccinating antigenin the construct. Such agents are useful for the identification,selection and purification of proteins useful in the invention.

In addition, the present invention contemplates the use of peptides thatare fused to the C-terminus of the antigen construct, particularly foruse in the selection and identification of the protein. Such peptidesinclude, but are not limited to, any synthetic or natural peptide, suchas a peptide tag (e.g., 6×His) or any other short epitope tag. Peptidesattached to the C-terminus of an antigen according to the invention canbe used with or without the addition of the N-terminal peptidesdiscussed above.

Finally, the present inventors describe herein several different novelfusion protein HCV antigens for use in a yeast-based vaccine thatprovide multiple (two or more) immunogenic domains from one or moreantigens within the same construct. An exemplary fusion proteincomprising multiple immunogenic domains is the fusion protein comprisingthe HCV NS3 and Core proteins, or immunogenic portions thereof, that isdescribed herein. Others are also described below.

As described above, NS3 and Core are abundantly expressed in infectedcells, are required for viral replication and contain epitopes that arerecognized by both CD4⁺ and CD8⁺ T cells in acute and chronic infection.An additional advantage of targeting these proteins, and particularlyboth proteins in a single vaccine, is the high degree of conservation atthe amino acid level. Both the Core and NS3 proteins are highlyconserved among HCV genotypes 1a and 1b, the HCV strains most prevalentin the U.S. (Table 1). The Core protein displays a 98% amino acididentity among strains 1a and 1b, and identities ranging from 86-95% forthe other five HCV genotypes are observed compared to the HCV 1a proteinsequence. The NS3 protein is also highly conserved among the differentHCV strains—a 92% amino acid identity exists between strains 1a and 1b,and identities range from 81-86% for the other HCV genotypes compared tothe HCV 1a protein sequence. The high degree of conservation of the Coreand NS3 proteins among the various HCV genotypes signals the essentialnature of specific overall protein domains for viral function. Onevaccine of the present invention, despite being a single product, wasdesigned to target two viral antigens, NS3 protease and Core protein.This approach can readily be expanded to incorporate the proteinsequences of other essential and conserved HCV viral proteins to resultin an even broader cellular immune response. Such additional fusionproteins and vaccines are exemplified herein.

The nucleic acid and amino acid sequence for HCV polyprotein genes andthe polyproteins encoded thereby are known in the art. For example, thenucleic acid sequence of the polyprotein gene for Hepatitis C Virusstrain H77 is described in Database Accession No. AF011753 (gi:2327074)and is represented herein by SEQ ID NO:19. SEQ ID NO:19 encodes the HCVstrain H77 polyprotein, which has an amino acid sequence representedherein by SEQ ID NO:20. Within SEQ ID NO:20, the HCV proteins comprisethe following positions: HCV Core (positions 1 to 191 of SEQ ID NO:20);HCV E1 envelope glycoprotein (positions 192 to 383 of SEQ ID NO:20); HCVE2 envelope glycoprotein (positions 384 to 746 of SEQ ID NO:20); HCV P7ion channel (positions 747 to 809 of SEQ ID NO:20); HCV NS2metalloprotease (positions 810 to 1026 of SEQ ID NO:20); HCV NS3protease/helicase (positions 1027 to 1657 of SEQ ID NO:20); HCV NS4a NS3protease cofactor (positions 1658 to 1711 of SEQ ID NO:20); HCV NS4b(positions 1712 to 1972 of SEQ ID NO:20); HCV NS5a (positions 1973 to2420 of SEQ ID NO:20); and HCV NS5b RNA-dependent RNA polymerase(positions 2421 to 3011 of SEQ ID NO:20). As discussed above, strains ofHCV display high amino acid identity (e.g., see Table 1). Therefore,using the guidance provided herein and the reference to the exemplaryHCV strain, one of skill in the art will readily be able to a variety ofHCV-based fusion proteins from any HCV strain for use in thecompositions and vaccines of the present invention.

It is clear that control and clearance of HCV requires both CD4⁺ andCD8⁺ T cells and that the lack of adequate cellular immunity isassociated with development of chronic infection. It is appealingtherefore, to propose that stimulation of existing but insufficientHCV-specific CD4⁺ and CD8⁺ T cells in chronically HCV infectedindividuals will have a therapeutic benefit. Without being bound bytheory, the present inventors believe that the ideal HCV immunotherapyconsists of a non-pathogenic vector that can deliver antigens into theMHC class I and class II antigen presentation pathways to stimulatepotent CD4⁺ and CD8⁺ T cell responses. This vector should also becapable of repeated administration, similar to other therapeuticproducts. The vaccine and compositions of the present invention areideally suited to these goals.

Some immunotherapeutic vaccine preparations known prior to the presentinvention consisted of purified viral proteins that are endocytosed bydendritic cells and macrophages (also referred to generally herein asantigen presenting cells or APCs). The proteins in the engulfed materialare digested into polypeptides (10-20 amino acids) which are bound toclass II MHC molecules in specialized endosomes in APCs. Thepeptide+class II MHC molecule complex is then expressed on the surfaceof the APC. An antigen-specific CD4⁺ helper T cell (T_(H)) binds to thecombination of class II MHC+peptide, becomes activated and produceslymphokines.

Soluble antigens that are administered extracellularly without adjuvantstend to stimulate type 2 helper T cells (T_(H)2), which producelymphokines that act on B cells leading to a humoral immune response.T_(H)2 responses tend to inhibit type 1 helper T cell (T_(H)1) responsesthat are important for induction of cell-mediated immunity. If the viralantigen being targeted is on the membrane of the infected cell,approaches that generate antibodies could have a therapeutic effect.However, if the viral antigen being targeted is found inside theinfected cell, antibody generally has little effect. In addition, andbecause of the bias towards a T_(H)2 response, CD8⁺ CTL are not normallyactivated in response to exogenously introduced protein antigens. IfCD8⁺ CTL are required for protection against chronic viral infection, itseems reasonable to postulate that approaches employing recombinantproteins may prove to be unsuccessful.

In contrast to extracellular antigens, CD8⁺ CTL are induced in responseto any antigen that is being synthesized by the cell to be targeted.These antigens are referred to as endogenous antigens. Viral proteinsbeing synthesized by infected cells are digested into peptides (8-10amino acids) by cytosolic proteasomes coupled with peptide delivery intothe endoplasmic reticulum. Proper folding of class I MHC molecules inthe endoplasmic reticulum is dependent on binding ofproteasome-generated peptides, prior to trafficking to the surface ofthe infected or tumor cell. CD8⁺ T cells respond to the combination ofMHC I receptor-peptide complexes and produce lymphokines including IFN-γwhich, in general, lead to a cell-mediated immune response, includingkilling of the infected cell.

CTL appear to require IL-2 and IL-12 in order to be effectivelyactivated. While CD8⁺ CTL can produce some IL-2, it is generallyaccepted that CD4⁺ T_(H)1 cells are the major sources of IL-2 forCTL-mediated responses. IL-12 is produced by dendritic cells andmacrophages. In addition, it is also clear that in order to obtainmaximal CTL activation, presentation of antigens by dendritic cells isrequired. Thus, as for CD4+T_(H)1 cells, CTL require interaction with anantigen presenting cell (APC) in order to become maximally activated andthen respond to virally-infected cells.

It was initially unclear how antigens being synthesized by avirally-infected cell could find their way into the class I MHC pathwayin dendritic cells, unless the dendritic cell itself became infected.However, recent data indicates that dendritic cells can recognizeinfected cells that become apoptotic as a result of infection and that“cross-priming” (delivery of exogenous antigens into the endogenousantigen presentation pathway) can occur such that some of the proteinsassociated with cells/particles engulfed by dendritic cells andmacrophages find their way into the class I MHC pathway (23). Inaddition, certain “danger” signals (described below) can enhance thisprocess (25).

Immune responses are initiated primarily by dendritic cells andmacrophages that take up foreign material from extracellular fluids. Amethod to increase the ability of these cells to adequately presentantigens should lead to an improved T cell-mediated cellular immuneresponse. In this regard, recombinant S. cerevisiae yeast exhibit theparticulate features of immunostimulatory complexes (ISCOMs) (26) withthe added advantage that richly glycosylated yeast possess naturaladjuvant-like properties and can be readily engineered to expressmultiple antigens (16, 27-29). S. cerevisiae yeast cells are avidlytaken up by professional antigen-presenting cells including macrophagesand dendritic cells. Yeast-associated proteins are efficiently presentedvia both class I and class II MHC leading to protective antigen-specificCTL-mediated immunity to tumor cells (16-17).

Dendritic cells and macrophages have a variety of receptors on theirsurface that act as microbial pattern recognition molecules; i.e., theyrecognize pathogens on the basis of differences in glycosylationpatterns, lipoproteins and nucleic acid composition. Hence, such antigenpresenting cells (APCs) have receptors for microbial mannoproteins,peptidoglycans, glucans, lipoproteins, double-stranded RNA and CpGisland-containing DNA (30-32). Engagement of these receptors results inwhat has been termed a “danger” signal leading to dendritic cellmaturation, activation, enhanced phagocytosis, and efficientpresentation of antigens that were associated with the engaging material(33).

In fact, dendritic cells and macrophages may have more receptors thatrecognize yeast than any other microbe. These receptors include TLR-2,TLR-4, TLR-6, CD14, Dectin-1, Dectin-2, DEC-205 and the mannose receptorfamily (30, 34). Uptake of zymosan, a crude Saccharomyces cerevisiaeyeast cell wall preparation, results in up-regulation of a multitude ofpro-inflammatory genes (35). The present inventors' data indicate thatuptake of whole yeast by mouse and human dendritic cells and macrophagesresults in upregulation of a variety of cell surface molecules includingadhesion molecules (ICAM-1, CD54), co-stimulatory molecules (B7-1, B7-2,CD80, CD86), and class I and class II MHC molecules, as well aspromoting the secretion of pro-inflammatory T_(H)1-type cytokines, suchas TNF-α, GM-CSF, interferon-γ, IL-2 and IL-12.

In addition to being able to interact directly with dendritic cells,yeast have a variety of other characteristics that make them an idealplatform for immunotherapy. First, multiple antigens may be engineeredfor expression within a single yeast strain (29), and these formulationsshare many advantages with DNA vaccines, including ease of constructionand the ability to target multiple antigens. Unlike DNA vaccines,yeast-based immunotherapeutic formulations do not require extensivepurification to remove potentially toxic contaminants. As will bedescribed in further detail below, the heterologous proteins expressedin recombinant yeast serve as antigens for potent CD8⁺ CTL-mediatedimmune responses in vitro and in vivo (16-17). In animal trials aspreventative, as well as therapeutic treatments, the yeast formulationwas successful at protecting and treating immunized animals from tumorgrowth (16-17). These results suggest that the vaccines of the presentinvention could be effective for eliciting broad-spectrum immuneresponses as an HCV immunotherapeutic.

In the present invention, the present inventors have generated a novelrecombinant yeast immunotherapeutic, also referred to herein as GI-5005,that expresses an HCVNS3-Core fusion protein under the control of aninducible promoter. Immunoblot analysis of GI-5005 cell lysates usingNS3- or Core-specific antibodies reveal a 47 kD protein. The GI-5005yeast produce greater than 5 μg of the HCV fusion protein per 10 millioncells. Injection of GI-5005 yeast in C57BL/6 and BALB/c mice resulted ininduction of potent NS3 and Core antigen-specific helper and cytotoxic Tcell immune responses as shown by lymphocyte proliferation, cytotoxicityand cytokine release assays. Mice that were vaccinated with GI-5000series yeast were protected from challenge with HCV antigen-expressingsyngeneic tumor cells. Immunogenicity and tumor protection results, aswell as results in a surrogate model of therapy are also presentedherein. Finally, a phase 1 trial in chronically HCV infected patientswill be described.

Vaccines and Compositions of the Invention

One embodiment of the present invention relates to a composition(vaccine) which can be used in a method to protect an animal against aHCV infection or disease resulting therefrom or to alleviate at leastone symptom resulting from the HCV infection. The composition orvaccine. The vaccine comprises: (a) a yeast vehicle; and (b) aheterologous fusion protein expressed by the yeast vehicle. As discussedabove, the invention includes several improved HCV fusion proteins foruse as antigens in the vaccines of the invention, wherein such vaccinesmay include yeast vehicles, although other vaccines that do not includeyeast vehicles are also contemplated by the present invention (seebelow). Specifically, the present invention provides new fusion proteinconstructs that stabilize the expression of the heterologous protein inthe yeast vehicle, prevent posttranslational modification of theexpressed heterologous protein, and/or that can be used as vaccinatingantigens in the absence of the yeast vehicle described herein (i.e., inconventional or other non-yeast-based vaccine compositions). The novelfusion proteins, in some embodiments, also provide a broad cellularimmune response by the use of multiple selected antigens in a singlevaccine. In conjunction with the yeast vehicle, these fusion proteinsare most typically expressed as recombinant proteins by the yeastvehicle (e.g., by an intact yeast or yeast spheroplast, which canoptionally be further processed to a yeast cytoplast, yeast ghost, oryeast membrane extract or fraction thereof), although it is anembodiment of the invention that one or much such fusion proteins couldbe loaded into a yeast vehicle or otherwise complexed or mixed with ayeast vehicle as described above to form a vaccine of the presentinvention.

One such fusion construct useful in the present invention is a fusionprotein that includes: (a) at least one HCV antigen (includingimmunogenic domains and epitopes of a full-length antigen, as well asvarious fusion proteins and multiple antigen constructs as describedelsewhere herein); and (b) a synthetic peptide.

In one embodiment, the synthetic peptide linked to the N-terminus of theHCV antigen, the peptide consisting of at least two amino acid residuesthat are heterologous to the HCV antigen, wherein the peptide stabilizesthe expression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein. Thesynthetic peptide and N-terminal portion of the antigen together form afusion protein that has the following requirements: (1) the amino acidresidue at position one of the fusion protein is a methionine (i.e., thefirst amino acid in the synthetic peptide is a methionine); (2) theamino acid residue at position two of the fusion protein is not aglycine or a proline (i.e., the second amino acid in the syntheticpeptide is not a glycine or a proline); (3) none of the amino acidresidues at positions 2-6 of the fusion protein is a methionine (i.e.,the amino acids at positions 2-6, whether part of the synthetic peptideor the protein, if the synthetic peptide is shorter than 6 amino acids,do not include a methionine); and (4) none of the amino acids atpositions 2-6 of the fusion protein is a lysine or an arginine (i.e.,the amino acids at positions 2-6, whether part of the synthetic peptideor the protein, if the synthetic peptide is shorter than 5 amino acids,do not include a lysine or an arginine). The synthetic peptide can be asshort as two amino acids, but is more preferably at least 2-6 aminoacids (including 3, 4, 5 amino acids), and can be longer than 6 aminoacids, in whole integers, up to about 200 amino acids.

In one embodiment, the peptide comprises an amino acid sequence ofM-X₂-X₃-X₄-X₅-X₆, wherein M is methionine; wherein X₂ is any amino acidexcept glycine, proline, lysine or arginine; wherein X₃ is any aminoacid except methionine, lysine or arginine; wherein X₄ is any amino acidexcept methionine, lysine or arginine; wherein X₅ is any amino acidexcept methionine, lysine or arginine; and wherein X₆ is any amino acidexcept methionine, lysine or arginine. In one embodiment, the X₆ residueis a proline. An exemplary synthetic sequence that enhances thestability of expression of an HCV antigen in a yeast cell and/orprevents post-translational modification of the protein in the yeastincludes the sequence M-A-D-E-A-P (SEQ ID NO:9). In addition to theenhanced stability of the expression product, the present inventorsbelieve that this fusion partner does not appear to negatively impactthe immune response against the vaccinating antigen in the construct. Inaddition, the synthetic fusion peptides can be designed to provide anepitope that can be recognized by a selection agent, such as anantibody.

In another embodiment of the invention, the nucleic acids that encodethe translation start site of a synthetic peptide used in the inventionare A-C-C-A-T-G-G in accordance with Kozak translation sequence rules,where the ATG in this sequence is the initial translation start site andencodes the methionine of M-A-D-E-A-P (SEQ ID NO:9).

It is to be understood that various embodiments of the invention asdescribed herein may also be combined. For example, in one aspect of theinvention, when the synthetic peptide is M-A-D-E-A-P (SEQ ID NO:9), thenucleic acids encoding the start site for this peptide can beA-C-C-A-T-G-G as described above. Various other combinations ofembodiments of the invention will be apparent to those of skill in theart.

Another specific embodiment of the present invention that is similar tothe embodiment above and that can include the limitations of theembodiment above (although this is not required) includes a vaccinecomprising: (iii) a peptide linked to the C-terminus of the HCV antigen,the peptide consisting of at least two amino acid residues that areheterologous to the HCV antigen, wherein the peptide stabilizes theexpression of the fusion protein in the yeast vehicle or preventsposttranslational modification of the expressed fusion protein. In oneexemplary aspect of the invention, the peptide comprises an amino acidsequence of E-D (Glu-Asp). Such a sequence works to counteracthydrophobicity.

According to the present invention, “heterologous amino acids” are asequence of amino acids that are not naturally found (i.e., not found innature, in vivo) flanking the specified amino acid sequence, or that arenot related to the function of the specified amino acid sequence, orthat would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the specified amino acidsequence as it occurs in the gene, if such nucleotides in the naturallyoccurring sequence were translated using standard codon usage for theorganism from which the given amino acid sequence is derived. Therefore,at least two amino acid residues that are heterologous to the HCVantigen are any two amino acid residues that are not naturally foundflanking the HCV antigen.

Another embodiment of the present invention relates to a composition(vaccine) that can be used for protecting an animal against HCVinfection or a symptom resulting from such infection comprising: (a) ayeast vehicle; and (b) a heterologous fusion protein expressed by theyeast vehicle. In one embodiment, the fusion protein comprises: (i) atleast one HCV antigen (including immunogenic domains and epitopes of afull-length antigen, as well as various fusion proteins and multipleantigen constructs as described elsewhere herein) that is fused to (ii)a yeast protein linked to the N-terminus of the HCV antigen, wherein theyeast protein consists of between about two and about 200 amino acids ofan endogenous yeast protein, wherein the yeast protein providessignificantly enhanced stability of the expression of the fusion proteinin the yeast vehicle or prevents posttranslational modification of theexpressed fusion protein by the yeast cells. In addition, the endogenousyeast antigen, as with the synthetic peptide, this fusion partner doesnot appear to negatively impact the immune response against thevaccinating antigen in the construct. This aspect of the invention maybe used in connection with other embodiments of the invention describedabove.

The endogenous yeast protein consists of between about two and about 200amino acids (or 22 kDa maximum) of an endogenous yeast protein, whereinthe yeast protein stabilizes the expression of the fusion protein in theyeast vehicle or prevents posttranslational modification of theexpressed fusion protein. Any suitable endogenous yeast protein can beused in this embodiment, and particularly preferred proteins include,but are not limited to, SUC2 (yeast invertase; which is a good candidatefor being able to express a protein both cytosolically and directing itinto the secretory pathway from the same promoter, but is dependent onthe carbon source in the medium); alpha factor signal leader sequence;SEC7; CPY; phosphoenolpyruvate carboxykinase PCK1, phosphoglycerokinasePGK and triose phosphate isomerase TPI gene products for theirrepressible expression in glucose and cytosolic localization; Cwp2p forits localization and retention in the cell wall; the heat shock proteinsSSA1, SSA3, SSA4, SSC1 and KAR2, whose expression is induced and whoseproteins are more thermostable upon exposure of cells to heat treatment;the mitochondrial protein CYC 1 for import into mitochondria; BUD genesfor localization at the yeast cell bud during the initial phase ofdaughter cell formation; ACT1 for anchoring onto actin bundles.

In one embodiment, the endogenous yeast protein/peptide or the syntheticpeptide used in fusion proteins herein comprise an antibody epitope foridentification and purification of the fusion protein. Antibodies mayalready be available that selectively bind to an endogenous antigen orcan be readily generated. Finally, if it is desired to direct a proteinto a particular cellular location (e.g., into the secretory pathway,into mitochondria, into the nucleus), then the construct can use theendogenous signals for the yeast protein to be sure that the cellularmachinery is optimized for that delivery system. Preferably, an antibodyis available or produced that selectively binds to the fusion partner.According to the present invention, the phrase “selectively binds to”refers to the ability of an antibody, antigen binding fragment orbinding partner of the present invention to preferentially bind tospecified proteins. More specifically, the phrase “selectively binds”refers to the specific binding of one protein to another (e.g., anantibody, fragment thereof, or binding partner to an antigen), whereinthe level of binding, as measured by any standard assay (e.g., animmunoassay), is statistically significantly higher than the backgroundcontrol for the assay. For example, when performing an immunoassay,controls typically include a reaction well/tube that contain antibody orantigen binding fragment alone (i.e., in the absence of antigen),wherein an amount of reactivity (e.g., non-specific binding to the well)by the antibody or antigen binding fragment thereof in the absence ofthe antigen is considered to be background. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.).

In one embodiment, a vaccine of the present invention can comprise apeptide linked to the C-terminus of the HCV antigen, wherein the peptideallows for recognition of the fusion protein by an antibody directedagainst the peptide. In one aspect, the peptide comprises an amino acidsequence of G-G-G-H-H-H-H-H-H (SEQ ID NO:10). This embodiment can beused alone or in conjunction with other aspects of the fusion proteinsdescribed above.

As discussed above, the fusion proteins used in the vaccines andcompositions of the invention include at least one HCV antigen forvaccinating an animal. The composition or vaccine can include, one, two,a few, several or a plurality of HCV antigens, including one or moreimmunogenic domains of one or more HCV antigens, as desired. Forexample, any fusion protein described herein can include at least aportion of any one or more HCV proteins selected from: HCV E1 envelopeglycoprotein, HCV E2 envelope glycoprotein, HCV P7 ion channel, HCV NS2metalloprotease, HCV NS3 protease/helicase, HCV NS4a NS3 proteasecofactor, HCV NS4b, HCV NS5a, HCV NS5b RNA-dependent RNA polymerase, andHCV Core sequence. In a preferred embodiment, a portion of an HCVprotein other than the HCV Core sequence is linked to at least a portionof an HCV Core sequence. In another aspect, the fusion protein comprisesat least one or more immunogenic domains of one or more HCV antigens.

According to the present invention, the general use herein of the term“antigen” refers: to any portion of a protein (peptide, partial protein,full-length protein), wherein the protein is naturally occurring orsynthetically derived, to a cellular composition (whole cell, celllysate or disrupted cells), to an organism (whole organism, lysate ordisrupted cells) or to a carbohydrate or other molecule, or a portionthereof, wherein the antigen elicits an antigen-specific immune response(humoral and/or cellular immune response), or alternatively acts as atoleragen, against the same or similar antigens that are encounteredwithin the cells and tissues of the animal to which the antigen isadministered.

In one embodiment of the present invention, when it is desirable tostimulate an immune response, the term “antigen” can be usedinterchangeably with the term “immunogen”, and is used herein todescribe a protein, peptide, cellular composition, organism or othermolecule which elicits a humoral and/or cellular immune response (i.e.,is antigenic), such that administration of the immunogen to an animal(e.g., via a vaccine of the present invention) mounts anantigen-specific immune response against the same or similar antigensthat are encountered within the tissues of the animal. Therefore, tovaccinate an animal against a particular antigen means, in oneembodiment, that an immune response is elicited against the antigen orimmunogenic or toleragenic portion thereof, as a result ofadministration of the antigen. Vaccination preferably results in aprotective or therapeutic effect, wherein subsequent exposure to theantigen (or a source of the antigen) elicits an immune response againstthe antigen (or source) that reduces or prevents a disease or conditionin the animal. The concept of vaccination is well known in the art. Theimmune response that is elicited by administration of a therapeuticcomposition of the present invention can be any detectable change in anyfacet of the immune response (e.g., cellular response, humoral response,cytokine production), as compared to in the absence of theadministration of the vaccine.

A “vaccinating antigen” can be an immunogen or a toleragen, but is anantigen used in a vaccine, where a biological response (elicitation ofan immune response, tolerance) is to be elicited against the vaccinatingantigen.

An immunogenic domain (portion, fragment, epitope) of a given antigencan be any portion of the antigen (i.e., a peptide fragment or subunitor an antibody epitope or other conformational epitope) that contains atleast one epitope that acts as an immunogen when administered to ananimal. For example, a single protein can contain multiple differentimmunogenic domains. Immunogenic domains need not be linear sequenceswithin a protein, in the case of a humoral response.

An epitope is defined herein as a single immunogenic site within a givenantigen that is sufficient to elicit an immune response, or a singletoleragenic site within a given antigen that is sufficient to suppress,delete or render inactive an immune response. Those of skill in the artwill recognize that T cell epitopes are different in size andcomposition from B cell epitopes, and that epitopes presented throughthe Class I MHC pathway differ from epitopes presented through the ClassII MHC pathway. Epitopes can be linear sequence or conformationalepitopes (conserved binding regions). depending on the type of immuneresponse. An antigen can be as small as a single epitope, or larger, andcan include multiple epitopes. As such, the size of an antigen can be assmall as about 5-12 amino acids (e.g., a peptide) and as large as: afull length protein, including a multimer and fusion proteins, chimericproteins, whole cells, whole microorganisms, or portions thereof (e.g.,lysates of whole cells or extracts of microorganisms). In addition,antigens can include carbohydrates, which can be loaded into a yeastvehicle or into a composition of the invention. It will be appreciatedthat in some embodiments (i.e., when the antigen is expressed by theyeast vehicle from a recombinant nucleic acid molecule), the antigen isa protein, fusion protein, chimeric protein, or fragment thereof, ratherthan an entire cell or microorganism. Preferred HCV fusion proteins ofthe invention are described herein.

In yet another embodiment of the invention, the HCV antigen portion ofthe vaccine is produced as a fusion protein comprising two or moreantigens. In one aspect, the fusion protein can include two or moreimmunogenic domains or two or more epitopes of one or more antigens(e.g., the HCV NS3 sequence and the HCV Core sequence described herein).Such a vaccine may provide antigen-specific immunization in a broadrange of patients. For example, a multiple domain fusion protein usefulin the present invention may have multiple domains, wherein each domainconsists of a peptide from a particular protein, the peptide consistingof at least 4 amino acid residues flanking either side of and includinga mutated amino acid that is found in the protein, wherein the mutationis associated with a particular disease or condition (e.g., HCVinfection).

In one embodiment of the present invention, any of the amino acidsequences described herein can be produced with from at least one, andup to about 20, additional heterologous amino acids flanking each of theC- and/or N-terminal ends of the specified amino acid sequence. Theresulting protein or polypeptide can be referred to as “consistingessentially of” the specified amino acid sequence. As discussed above,according to the present invention, the heterologous amino acids are asequence of amino acids that are not naturally found (i.e., not found innature, in vivo) flanking the specified amino acid sequence, or that arenot related to the function of the specified amino acid sequence, orthat would not be encoded by the nucleotides that flank the naturallyoccurring nucleic acid sequence encoding the specified amino acidsequence as it occurs in the gene, if such nucleotides in the naturallyoccurring sequence were translated using standard codon usage for theorganism from which the given amino acid sequence is derived. Similarly,the phrase “consisting essentially of”, when used with reference to anucleic acid sequence herein, refers to a nucleic acid sequence encodinga specified amino acid sequence that can be flanked by from at leastone, and up to as many as about 60, additional heterologous nucleotidesat each of the 5′ and/or the 3′ end of the nucleic acid sequenceencoding the specified amino acid sequence. The heterologous nucleotidesare not naturally found (i.e., not found in nature, in vivo) flankingthe nucleic acid sequence encoding the specified amino acid sequence asit occurs in the natural gene or do not encode a protein that impartsany additional function to the protein or changes the function of theprotein having the specified amino acid sequence.

In one preferred aspect of the invention, the HCV antigen is an HCVprotein consisting of HCV NS3 protease and Core sequence. In anotheraspect, the HCV antigen consists of an HCV NS3 protein lacking thecatalytic domain of the natural NS3 protein which is linked to HCV Coresequence. In another aspect, the HCV antigen consists of the 262 aminoacids of HCV NS3 following the initial N-terminal 88 amino acids of thenatural NS3 protein (i.e., positions 89-350 of HCV NS3; SEQ ID NO:20)linked to HCV Core sequence. In one aspect, the HCV Core sequence lacksthe hydrophobic C-terminal sequence. In another aspect, the HCV Coresequence lacks the C-terminal two amino acids, glutamate and aspartate.In a preferred aspect, the HCV Core sequence consists of amino acidpositions 2 through 140 of the natural HCV Core sequence.

An example of such a vaccine is described in Example 1. In thisembodiment, a yeast (e.g., Saccharomyces cerevisiae) was engineered toexpress a HCV NS3-Core fusion protein under the control of thecopper-inducible promoter, CUP1. The fusion protein is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus (HCV polyprotein (SEQ ID NO:20) numbering in parentheses,with the amino acid sequence of the fusion protein being representedherein by SEQ ID NO:2): 1) the sequence MADEAP (SEQ ID NO:9) to impartresistance to proteasomal degradation (positions 1 to 6 of SEQ ID NO:2);2) amino acids 89 to 350 of (1115 to 1376 of SEQ ID NO:20) of the HCVNS3 protease protein (positions 6 to 268 of SEQ ID NO:2); 3) a singlethreonine amino acid residue introduced in cloning (position 269 of SEQID NO:2); 4) amino acids 2 to 140 (2 to 140 of SEQ ID NO:20) of the HCVCore protein (positions 270 to 408 of SEQ ID NO:2); and 5) the sequenceE-D to increase the hydrophilicity of the Core variant (positions 409 to410 of SEQ ID NO:2). A nucleic acid sequence encoding the fusion proteinof SEQ ID NO:2 is represented herein by SEQ ID NO:1.

In another preferred aspect of the invention, the HCV antigen is aninactivated full-length HCV NS3 that is part of a fusion proteinaccording to the invention. An example of such a vaccine is described inExample 2. In this embodiment, a yeast (e.g., Saccharomyces cerevisiae)was engineered to express an inactivated full-length HCV NS3 fusionprotein under the control of the copper-inducible promoter, CUP1. Thefusion protein comprising the full-length HCV NS3 is a singlepolypeptide with the following sequence elements fused in frame from N-to C-terminus (HCV polyprotein numbering in parentheses, with the aminoacid sequence of the fusion protein being represented herein by SEQ IDNO:4): 1) the sequence MADEAP (SEQ ID NO:9) to impart resistance toproteasomal degradation (positions 1 to 6 of SEQ ID NO:4); and 2) aminoacids 1 to 631 (1027 to 1657 of SEQ ID NO:20) of the HCV NS3 proteaseprotein (positions 7 to 637 of SEQ ID NO:4) (note that the amino acid atHCV polypeptide residue 1165 has been changed from a serine to analanine in order to inactivate the proteolytic activity). A nucleic acidsequence encoding the fusion protein of SEQ ID NO:4 is representedherein by SEQ ID NO:3.

In another preferred aspect of the invention, the yeast vaccinecomprises a truncated HCV E1-E2 fusion protein. An example of such avaccine is described in Example 3. In this embodiment, a yeast (e.g.,Saccharomyces cerevisiae) is engineered to express an E1-E2 fusionprotein as a single polypeptide having the following sequence elementsfused in frame from N- to C-terminus (HCV polyprotein numbering inparentheses, where the amino acid sequence of the fusion protein isrepresented herein by SEQ ID NO:6): 1) The sequence MADEAP (SEQ ID NO:9)to impart resistance to proteasomal degradation (positions 1 to 6 of SEQID NO:6); 2) amino acids 1 to 156 (192 to 347 of SEQ ID NO:20) of HCVprotein E1 (positions 7 to 162 of SEQ ID NO:6); and 3) amino acids 1 to334 (384 to 717 of SEQ ID NO:20) of HCV protein E2 (positions 163 to 446of SEQ ID NO:6). It is noted that in this particular fusion protein, 36C-terminal hydrophobic amino acids of E1 and 29 C-terminal hydrophobicamino acids of E2 were omitted from the fusion protein to promotecytoplasmic accumulation in yeast. A nucleic acid sequence encoding thefusion protein of SEQ ID NO:6 is represented herein by SEQ ID NO:5.

In yet another preferred aspect of the invention, the yeast vaccinecomprises a transmembrane (TM) domain-deleted HCV NS4b fusion protein.An example of such vaccine is described in Example 4. The fusion proteinis a single polypeptide with the following sequence elements arranged intandem, in frame, from N- to C-terminus (polyprotein numbering inparentheses, with the amino acid sequence of the fusion protein beingrepresented herein by SEQ ID NO:8): 1) The sequence MADEAP (SEQ ID NO:9)to impart resistance to proteosomal degradation (positions 1 to 6 of SEQID NO:8); 2) amino acids 1 to 69 (1712 to 1780 of SEQ ID NO:20) of HCVprotein NS4b (positions 7 to 75 of SEQ ID NO:8); and 3) amino acids 177to 261 (1888 to 1972 of SEQ ID NO:20) of HCV protein NS4b (positions 76to 160 of SEQ ID NO:8). A 107 amino acid region corresponding to NS4bamino acids 70 to 176 (1781 to 1887 of SEQ ID NO:20) that containsmultiple membrane spanning domains was omitted to promote cytoplasmicaccumulation in yeast. A nucleic acid sequence encoding the fusionprotein of SEQ ID NO:8 is represented herein by SEQ ID NO:7.

In yet another preferred aspect of the invention, the yeast vaccinecomprises a Core-E1-E2 fusion protein. The fusion protein is a singlepolypeptide with the following sequence elements arranged in tandem, inframe, from N- to C-terminus (polyprotein numbering in parentheses, withthe amino acid sequence of the fusion protein being represented hereinby SEQ ID NO:12): 1) The sequence MADEAP (SEQ ID NO:9) to impartresistance to proteosomal degradation (positions 1-6 of SEQ ID NO:12);and 2) amino acids 1 to 746 (2 to 746 of SEQ ID NO:20) of unmodified HCVpolyprotein encoding full-length Core, E1, and E2 proteins (positions 7to 751 of SEQ ID NO:12: Core spanning from position 7 to 196; E1spanning from positions 197 to 387; and E2 spanning from positions 388to 751). A nucleic acid sequence encoding the fusion protein of SEQ IDNO:12 is represented herein by SEQ ID NO:11.

In another preferred aspect of the invention, the yeast vaccinecomprises a Core-E1-E2 fusion protein with transmembrane domainsdeleted. The fusion protein is a single polypeptide with the followingsequence elements fused in frame from N- to C-terminus (polyproteinnumbering in parentheses, with the amino acid sequence of the fusionprotein being represented herein by SEQ ID NO:14): 1) The sequenceMADEAP (SEQ ID NO:9) to impart resistance to proteasomal degradation, 2)amino acids 2 to 140 (2 to 140 of SEQ ID NO:20) of HCV Core protein(positions 7 to 145 of SEQ ID NO:14), 3) amino acids 1 to 156 (192 to347 of SEQ ID NO:20) of HCV protein E1 (positions 146 to 301 of SEQ IDNO:14), and 4) amino acids 1 to 334 (384 to 717 of SEQ ID NO:20) of HCVprotein E2 (positions 302 to 635 of SEQ ID NO:14). The 51 C-terminalhydrophobic amino acids of Core protein, the 36 C-terminal hydrophobicamino acids of E1 and the 29 C-terminal hydrophobic amino acids of E2were omitted from the fusion protein to promote cytoplasmic accumulationin yeast. A nucleic acid sequence encoding the fusion protein of SEQ IDNO:14 is represented herein by SEQ ID NO:13.

In yet another preferred aspect of the invention, the yeast vaccinecomprises an NS3-NS4a-NS4b fusion protein wherein the NS3 protease isinactivated and the NS4b lacks a transmembrane domain. The NS3-NS4a-NS4bfusion protein is a single polypeptide with the following sequenceelements fused in frame from N- to C-terminus (polyprotein numbering inparentheses, with the amino acid sequence of the fusion protein beingrepresented herein by SEQ ID NO:16): 1) The sequence MADEAP (SEQ IDNO:9) to impart resistance to proteasomal degradation (positions 1 to 6of SEQ ID NO:16); 2) amino acids 1 to 631 (1027 to 1657 of SEQ ID NO:20)corresponding to full-length HCV NS3 protein (note: Serine 139 (position1165, with respect to SEQ ID NO:20) is changed to alanine to inactivatethe proteolytic potential of NS3) (positions 7 to 634 of SEQ ID NO:16);3) amino acids 1 to 54 (1658 to 1711 of SEQ ID NO:20) of NS4a protein(positions 635 to 691 of SEQ ID NO:16); 4) amino acids 1 to 69 (1712 to1780 of SEQ ID NO:20) of HCV protein NS4b (positions 692 to 776 of SEQID NO:16); and 5) amino acids 177 to 261 (1888 to 1972 of SEQ ID NO:20)of HCV protein NS4b (positions 777 to 845 of SEQ ID NO:16). A 107 aminoacid region corresponding to NS4b amino acids 70 to 176 (1781 to 1887 ofSEQ ID NO:20) that contains multiple membrane spanning domains wasomitted to promote cytoplasmic accumulation in yeast. A nucleic acidsequence encoding the fusion protein of SEQ ID NO:16 is representedherein by SEQ ID NO:15.

In another preferred aspect of the invention, the yeast vaccinecomprises a NS5a-NS5b fusion protein with an inactivating deletion ofNS5b C-terminus. This NS5a-NS5b fusion protein is a single polypeptidewith the following sequence elements fused in frame from N- toC-terminus (polyprotein numbering in parentheses, with the amino acidsequence of the fusion protein being represented herein by SEQ IDNO:18): 1) The sequence MADEAP (SEQ ID NO:9) to impart resistance toproteasomal degradation (positions 1 to 6 of SEQ ID NO:18); 2) theentirety of NS5a protein corresponding to amino acids 1 to 448 (1973 to2420 of SEQ ID NO:20) (positions 7 to 454 of SEQ ID NO:18); and 3) aminoacids 1 to 539 (2421 to 2959 of SEQ ID NO:20) of NS5b (positions 455 to993 of SEQ ID NO:18). The 52 C-terminal residues that are required forthe activity of NS5b in HCV replication were deleted to inactivate theprotein. A nucleic acid sequence encoding the fusion protein of SEQ IDNO:18 is represented herein by SEQ ID NO:17.

According to the present invention, any of the fusion proteins describedherein can comprise a peptide linked to the N-terminus of the fusionprotein that consists of at least 2-6 amino acid residues that areheterologous to the HCV antigen. In one aspect, the peptide comprises anamino acid sequence of M-X₂-X₃-X₄-X₅-X₆, wherein X₂ is any amino acidexcept glycine, proline, lysine or arginine; wherein X₃ is any aminoacid except methionine, lysine or arginine; wherein X₄ is any amino acidexcept methionine, lysine or arginine; wherein X₅ is any amino acidexcept methionine, lysine or arginine; and wherein X₆ is any amino acidexcept methionine. In one aspect, X₆ is a proline. In another aspect,the peptide comprises an amino acid sequence of M-A-D-E-A-P (SEQ IDNO:9).

In a particular aspect of the invention, the above-described fusionprotein contains a heterologous linker sequence between two HCV proteins(e.g., the HCV NS3 sequence and the HCV Core sequence). In a preferredembodiment, the heterologous linker sequence consists of a singleheterologous amino acid residue. In a more preferred embodiment, theheterologous linker sequence consists of a single threonine residue.

In any of the above-described compositions (e.g., vaccines) of thepresent invention, the following aspects related to the yeast vehicleare included in the invention. In one embodiment, yeast vehicle isselected from the group consisting of a whole yeast, a yeastspheroplast, a yeast cytoplast, a yeast ghost, and a subcellular yeastmembrane extract or fraction thereof. In one aspect, a yeast cell oryeast spheroplast used to prepare the yeast vehicle was transformed witha recombinant nucleic acid molecule encoding the antigen(s) such thatthe antigen is recombinantly expressed by the yeast cell or yeastspheroplast. In this aspect, the yeast cell or yeast spheroplast thatrecombinantly expresses the antigen(s) is used to produce a yeastvehicle comprising a yeast cytoplast, a yeast ghost, or a subcellularyeast membrane extract or fraction thereof. In one aspect, the yeastvehicle is from a non-pathogenic yeast. In another aspect, the yeastvehicle is from a yeast selected from the group consisting of:Saccharomyces, Schizosaccharomyces, Kluveromyces, Hansenula, Candida andPichia. In one aspect, the Saccharomyces is S. cerevisiae.

In general, the yeast vehicle and antigen can be associated by anytechnique described herein. In one aspect, the yeast vehicle was loadedintracellularly with the HCV antigen. In another aspect, the HCV antigenwas covalently or non-covalently attached to the yeast vehicle. In yetanother aspect, the yeast vehicle and the HCV antigen were associated bymixing. In another aspect, the antigen is expressed recombinantly by theyeast vehicle or by the yeast cell or yeast spheroplast from which theyeast vehicle was derived.

More specifically, according to the present invention, a yeast vehicleis any yeast cell (e.g., a whole or intact cell) or a derivative thereof(see below) that can be used in conjunction with an antigen in a vaccineor therapeutic composition of the invention, or as an adjuvant. Theyeast vehicle can therefore include, but is not limited to, a liveintact yeast microorganism (i.e., a yeast cell having all its componentsincluding a cell wall), a killed (dead) intact yeast microorganism, orderivatives thereof including: a yeast spheroplast (i.e., a yeast celllacking a cell wall), a yeast cytoplast (i.e., a yeast cell lacking acell wall and nucleus), a yeast ghost (i.e., a yeast cell lacking a cellwall, nucleus and cytoplasm), or a subcellular yeast membrane extract orfraction thereof (also referred to previously as a subcellular yeastparticle).

Yeast spheroplasts are typically produced by enzymatic digestion of theyeast cell wall. Such a method is described, for example, in Franzusoffet al., 1991, Meth. Enzymol. 194, 662-674., incorporated herein byreference in its entirety. Yeast cytoplasts are typically produced byenucleation of yeast cells. Such a method is described, for example, inCoon, 1978, Natl. Cancer Inst. Monogr. 48, 45-55 incorporated herein byreference in its entirety. Yeast ghosts are typically produced byresealing a permeabilized or lysed cell and can, but need not, containat least some of the organelles of that cell. Such a method isdescribed, for example, in Franzusoff et al., 1983, J. Biol. Chem. 258,3608-3614 and Bussey et al., 1979, Biochim. Biophys. Acta 553, 185-196,each of which is incorporated herein by reference in its entirety. Asubcellular yeast membrane extract or fraction thereof refers to a yeastmembrane that lacks a natural nucleus or cytoplasm. The particle can beof any size, including sizes ranging from the size of a natural yeastmembrane to microparticles produced by sonication or other membranedisruption methods known to those skilled in the art, followed byresealing. A method for producing subcellular yeast membrane extracts isdescribed, for example, in Franzusoff et al., 1991, Meth. Enzymol. 194,662-674. One may also use fractions of yeast membrane extracts thatcontain yeast membrane portions and, when the antigen was expressedrecombinantly by the yeast prior to preparation of the yeast membraneextract, the antigen of interest.

Any yeast strain can be used to produce a yeast vehicle of the presentinvention. Yeast are unicellular microorganisms that belong to one ofthree classes: Ascomycetes, Basidiomycetes and Fungi Imperfecti. Whilepathogenic yeast strains, or nonpathogenic mutants thereof can be usedin accordance with the present invention, nonpathogenic yeast strainsare preferred. Preferred genera of yeast strains include Saccharomyces,Candida (which can be pathogenic), Cryptococcus, Hansenula,Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia,with Saccharomyces, Candida, Hansenula, Pichia and Schizosaccharomycesbeing more preferred, and with Saccharomyces being particularlypreferred. Preferred species of yeast strains include Saccharomycescerevisiae, Saccharomyces carlsbergensis, Candida albicans, Candidakefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcusneoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromycesfragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. lactis,Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, andYarrowia lipolytica. It is to be appreciated that a number of thesespecies include a variety of subspecies, types, subtypes, etc. that aremeant to be included within the aforementioned species. More preferredyeast species include S. cerevisiae, C. albicans, H. polymorpha, P.pastoris and S. pombe. S. cerevisiae is particularly preferred due to itbeing relatively easy to manipulate and being “Generally Recognized AsSafe” or “GRAS” for use as food additives (GRAS, FDA proposed Rule62FR18938, Apr. 17, 1997). One embodiment of the present invention is ayeast strain that is capable of replicating plasmids to a particularlyhigh copy number, such as a S. cerevisiae cir^(o) strain.

In one embodiment, a preferred yeast vehicle of the present invention iscapable of fusing with the cell type to which the yeast vehicle andantigen is being delivered, such as a dendritic cell or macrophage,thereby effecting particularly efficient delivery of the yeast vehicle,and in many embodiments, the antigen(s), to the cell type. As usedherein, fusion of a yeast vehicle with a targeted cell type refers tothe ability of the yeast cell membrane, or particle thereof, to fusewith the membrane of the targeted cell type (e.g., dendritic cell ormacrophage), leading to syncytia formation. As used herein, a syncytiumis a multinucleate mass of protoplasm produced by the merging of cells.A number of viral surface proteins (including those of immunodeficiencyviruses such as HIV, influenza virus, poliovirus and adenovirus) andother fusogens (such as those involved in fusions between eggs andsperm) have been shown to be able to effect fusion between two membranes(i.e., between viral and mammalian cell membranes or between mammaliancell membranes). For example, a yeast vehicle that produces an HIVgp120/gp41 heterologous antigen on its surface is capable of fusing witha CD4+ T-lymphocyte. It is noted, however, that incorporation of atargeting moiety into the yeast vehicle, while it may be desirable undersome circumstances, is not necessary. The present inventors havepreviously shown that yeast vehicles of the present invention arereadily taken up by dendritic cells (as well as other cells, such asmacrophages).

Yeast vehicles can be formulated into compositions of the presentinvention, including preparations to be administered to a patientdirectly or first loaded into a carrier such as a dendritic cell, usinga number of techniques known to those skilled in the art. For example,yeast vehicles can be dried by lyophilization. Formulations comprisingyeast vehicles can also be prepared by packing yeast in a cake or atablet, such as is done for yeast used in baking or brewing operations.In addition, prior to loading into a dendritic cell, or other type ofadministration with an antigen, yeast vehicles can also be mixed with apharmaceutically acceptable excipient, such as an isotonic buffer thatis tolerated by the host cell. Examples of such excipients includewater, saline, Ringer's solution, dextrose solution, Hank's solution,and other aqueous physiologically balanced salt solutions. Nonaqueousvehicles, such as fixed oils, sesame oil, ethyl oleate, or triglyceridesmay also be used. Other useful formulations include suspensionscontaining viscosity-enhancing agents, such as sodiumcarboxymethylcellulose, sorbitol, glycerol or dextran. Excipients canalso contain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise, for example, dextrose, human serum albumin, and/orpreservatives to which sterile water or saline can be added prior toadministration.

According to the present invention, the term “yeast vehicle-antigencomplex” or “yeast-antigen complex” is used generically to describe anyassociation of a yeast vehicle with an antigen. Such associationincludes expression of the antigen by the yeast (a recombinant yeast),introduction of an antigen into a yeast, physical attachment of theantigen to the yeast, and mixing of the yeast and antigen together, suchas in a buffer or other solution or formulation. These types ofcomplexes are described in detail below.

In one embodiment, a yeast cell used to prepare the yeast vehicle istransformed with a heterologous nucleic acid molecule encoding theantigen such that the antigen is expressed by the yeast cell. Such ayeast is also referred to herein as a recombinant yeast or a recombinantyeast vehicle. The yeast cell can then be loaded into the dendritic cellas an intact cell, or the yeast cell can be killed, or it can bederivatized such as by formation of yeast spheroplasts, cytoplasts,ghosts, or subcellular particles, any of which is followed by loading ofthe derivative into the dendritic cell. Yeast spheroplasts can also bedirectly transfected with a recombinant nucleic acid molecule (e.g., thespheroplast is produced from a whole yeast, and then transfected) inorder to produce a recombinant spheroplast that expresses an antigen.

According to the present invention, an isolated nucleic acid molecule ornucleic acid sequence, is a nucleic acid molecule or sequence that hasbeen removed from its natural milieu. As such, “isolated” does notnecessarily reflect the extent to which the nucleic acid molecule hasbeen purified. An isolated nucleic acid molecule useful for transfectingyeast vehicles include DNA, RNA, or derivatives of either DNA or RNA. Anisolated nucleic acid molecule can be double stranded or singlestranded. An isolated nucleic acid molecule useful in the presentinvention includes nucleic acid molecules that encode a protein or afragment thereof, as long as the fragment contains at least one epitopeuseful in a composition of the present invention.

Nucleic acid molecules transformed into yeast vehicles of the presentinvention can include nucleic acid sequences encoding one or moreproteins, or portions (fragments, domains, conformational epitopes)thereof. Such nucleic acid molecules can comprise partial or entirecoding regions, regulatory regions, or combinations thereof. Oneadvantage of yeast strains is their ability to carry a number of nucleicacid molecules and of being capable of producing a number ofheterologous proteins. A preferred number of antigens to be produced bya yeast vehicle of the present invention is any number of antigens thatcan be reasonably produced by a yeast vehicle, and typically ranges fromat least one to at least about 5 or more, with from about 2 to about 5heterologous antigens being more preferred.

A peptide or protein encoded by a nucleic acid molecule within a yeastvehicle can be a full-length protein, or can be a functionallyequivalent protein in which amino acids have been deleted (e.g., atruncated version of the protein), inserted, inverted, substitutedand/or derivatized (e.g., acetylated, glycosylated, phosphorylated,tethered by a glycerophosphatidyl inositol (GPI) anchor) such that themodified protein has a biological function substantially similar to thatof the natural protein (or which has enhanced or inhibited function ascompared to the natural protein, if desired). Modifications can beaccomplished by techniques known in the art including, but not limitedto, direct modifications to the protein or modifications to the nucleicacid sequence encoding the protein using, for example, classic orrecombinant DNA techniques to effect random or targeted mutagenesis.Functionally equivalent proteins can be selected using assays thatmeasure the biological activity of the protein. Preferred HCV antigensare discussed above.

Expression of an antigen in a yeast vehicle of the present invention isaccomplished using techniques known to those skilled in the art.Briefly, a nucleic acid molecule encoding at least one desired antigenis inserted into an expression vector in such a manner that the nucleicacid molecule is operatively linked to a transcription control sequencein order to be capable of effecting either constitutive or regulatedexpression of the nucleic acid molecule when transformed into a hostyeast cell. Nucleic acid molecules encoding one or more antigens can beon one or more expression vectors operatively linked to one or moretranscription control sequences.

In a recombinant molecule of the present invention, nucleic acidmolecules are operatively linked to expression vectors containingregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the yeast cell and thatcontrol the expression of nucleic acid molecules. In particular,recombinant molecules of the present invention include nucleic acidmolecules that are operatively linked to one or more transcriptioncontrol sequences. The phrase “operatively linked” refers to linking anucleic acid molecule to a transcription control sequence in a mannersuch that the molecule is able to be expressed when transfected (i.e.,transformed, transduced or transfected) into a host cell.

Transcription control sequences, which can control the amount of proteinproduced, include sequences which control the initiation, elongation,and termination of transcription. Particularly important transcriptioncontrol sequences are those which control transcription initiation, suchas promoter and upstream activation sequences. Any suitable yeastpromoter can be used in the present invention and a variety of suchpromoters are known to those skilled in the art. Preferred promoters forexpression in Saccharomyces cerevisiae include, but are not limited to,promoters of genes encoding the following yeast proteins: alcoholdehydrogenase I (ADH1) or II (ADH2), CUP1, phosphoglycerate kinase(PGK), triose phosphate isomerase (TPI), glyceraldehyde-3-phosphatedehydrogenase (GAPDH; also referred to as TDH3, for triose phosphatedehydrogenase), galactokinase (GAL1), galactose-1-phosphateuridyl-transferase (GAL7), UDP-galactose epimerase (GAL10), cytochromec₁ (CYC1), Sec7 protein (SECT) and acid phosphatase (PHO5), with hybridpromoters such as ADH2/GAPDH and CYC1/GAL10 promoters being morepreferred, and the ADH2/GAPDH promoter, which is induced when glucoseconcentrations in the cell are low (e.g., about 0.1 to about 0.2percent), being even more preferred. Likewise, a number of upstreamactivation sequences (UASs), also referred to as enhancers, are known.Preferred upstream activation sequences for expression in Saccharomycescerevisiae include, but are not limited to, the UASs of genes encodingthe following proteins: PCK1, TPI, TDH3, CYC1, ADH1, ADH2, SUC2, GALL,GAL7 and GAL10, as well as other UASs activated by the GAL4 geneproduct, with the ADH2 UAS being particularly preferred. Since the ADH2UAS is activated by the ADR1 gene product, it is preferable tooverexpress the ADR1 gene when a heterologous gene is operatively linkedto the ADH2 UAS. Preferred transcription termination sequences forexpression in Saccharomyces cerevisiae include the termination sequencesof the α-factor, GAPDH, and CYC1 genes.

Preferred transcription control sequences to express genes inmethyltrophic yeast include the transcription control regions of thegenes encoding alcohol oxidase and formate dehydrogenase.

Transfection of a nucleic acid molecule into a yeast cell according tothe present invention can be accomplished by any method by which anucleic acid molecule administered into the cell and includes, but isnot limited to, diffusion, active transport, bath sonication,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. Transfected nucleic acid molecules can be integrated into ayeast chromosome or maintained on extrachromosomal vectors usingtechniques known to those skilled in the art. Examples of yeast vehiclescarrying such nucleic acid molecules are disclosed in detail herein. Asdiscussed above, yeast cytoplast, yeast ghost, and subcellular yeastmembrane extract or fractions thereof can also be produced recombinantlyby transfecting intact yeast microorganisms or yeast spheroplasts withdesired nucleic acid molecules, producing the antigen therein, and thenfurther manipulating the microorganisms or spheroplasts using techniquesknown to those skilled in the art to produce cytoplast, ghost orsubcellular yeast membrane extract or fractions thereof containingdesired antigens.

Effective conditions for the production of recombinant yeast vehiclesand expression of the antigen by the yeast vehicle include an effectivemedium in which a yeast strain can be cultured. An effective medium istypically an aqueous medium comprising assimilable carbohydrate,nitrogen and phosphate sources, as well as appropriate salts, minerals,metals and other nutrients, such as vitamins and growth factors. Themedium may comprise complex nutrients or may be a defined minimalmedium. Yeast strains of the present invention can be cultured in avariety of containers, including, but not limited to, bioreactors,Erlenmeyer flasks, test tubes, microtiter dishes, and petri plates.Culturing is carried out at a temperature, pH and oxygen contentappropriate for the yeast strain. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art (see, forexample, Guthrie et al. (eds.), 1991, Methods in Enzymology, vol. 194,Academic Press, San Diego).

In one embodiment of the present invention, as an alternative toexpression of an antigen recombinantly in the yeast vehicle, a yeastvehicle is loaded intracellularly with the protein or peptide antigen,or with carbohydrates or other molecules that serve as an antigen.Subsequently, the yeast vehicle, which now contains the antigenintracellularly, can be administered to the patient or loaded into acarrier such as a dendritic cell (described below). As used herein, apeptide comprises an amino acid sequence of less than or equal to about30-50 amino acids, while a protein comprises an amino acid sequence ofmore than about 30-50 amino acids; proteins can be multimeric. A proteinor peptide useful as an antigen can be as small as a T cell epitope(i.e., greater than 5 amino acids in length) and any suitable sizegreater than that which comprises multiple epitopes, protein fragments,full-length proteins, chimeric proteins or fusion proteins. Peptides andproteins can be derivatized either naturally or synthetically; suchmodifications can include, but are not limited to, glycosylation,phosphorylation, acetylation, myristylation, prenylation,palmitoylation, amidation and/or addition of glycerophosphatidylinositol. Peptides and proteins can be inserted directly into yeastvehicles of the present invention by techniques known to those skilledin the art, such as by diffusion, active transport, liposome fusion,electroporation, phagocytosis, freeze-thaw cycles and bath sonication.Yeast vehicles that can be directly loaded with peptides, proteins,carbohydrates, or other molecules include intact yeast, as well asspheroplasts, ghosts or cytoplasts, which can be loaded with antigensafter production, but before loading into dendritic cells.Alternatively, intact yeast can be loaded with the antigen, and thenspheroplasts, ghosts, cytoplasts, or subcellular particles can beprepared therefrom. Any number of antigens can be loaded into a yeastvehicle in this embodiment, from at least 1, 2, 3, 4 or any wholeinteger up to hundreds or thousands of antigens, such as would beprovided by the loading of a microorganism, by the loading of amammalian tumor cell, or portions thereof, for example.

In another embodiment of the present invention, an antigen is physicallyattached to the yeast vehicle. Physical attachment of the antigen to theyeast vehicle can be accomplished by any method suitable in the art,including covalent and non-covalent association methods which include,but are not limited to, chemically crosslinking the antigen to the outersurface of the yeast vehicle or biologically linking the antigen to theouter surface of the yeast vehicle, such as by using an antibody orother binding partner. Chemical cross-linking can be achieved, forexample, by methods including glutaraldehyde linkage, photoaffinitylabeling, treatment with carbodiimides, treatment with chemicals capableof linking di-sulfide bonds, and treatment with other cross-linkingchemicals standard in the art. Alternatively, a chemical can becontacted with the yeast vehicle that alters the charge of the lipidbilayer of yeast membrane or the composition of the cell wall so thatthe outer surface of the yeast is more likely to fuse or bind toantigens having particular charge characteristics. Targeting agents suchas antibodies, binding peptides, soluble receptors, and other ligandsmay also be incorporated into an antigen as a fusion protein orotherwise associated with an antigen for binding of the antigen to theyeast vehicle.

In yet another embodiment, the yeast vehicle and the antigen areassociated with each other by a more passive, non-specific ornon-covalent binding mechanism, such as by gently mixing the yeastvehicle and the antigen together in a buffer or other suitableformulation.

In one embodiment of the invention, the yeast vehicle and the antigenare both loaded intracellularly into a carrier such as a dendritic cellor macrophage to form the therapeutic composition or vaccine of thepresent invention. Alternatively, an antigen of the invention (i.e., anovel HCV fusion protein of the invention) can be loaded into adendritic cell in the absence of the yeast vehicle. Various forms inwhich the loading of both components can be accomplished are discussedin detail below. As used herein, the term “loaded” and derivativesthereof refer to the insertion, introduction, or entry of a component(e.g., the yeast vehicle and/or antigen) into a cell (e.g., a dendriticcell). To load a component intracellularly refers to the insertion orintroduction of the component to an intracellular compartment of thecell (e.g., through the plasma membrane and at a minimum, into thecytoplasm, a phagosome, a lysosome, or some intracellular space of thecell). To load a component into a cell references any technique by whichthe component is either forced to enter the cell (e.g., byelectroporation) or is placed in an environment (e.g., in contact withor near to a cell) where the component will be substantially likely toenter the cell by some process (e.g., phagocytosis). Loading techniquesinclude, but are not limited to: diffusion, active transport, liposomefusion, electroporation, phagocytosis, and bath sonication. In apreferred embodiment, passive mechanisms for loading a dendritic cellwith the yeast vehicle and/or antigen are used, such passive mechanismsincluding phagocytosis of the yeast vehicle and/or antigen by thedendritic cell.

It is noted that any of the above-described HCV fusion proteins can beprovided in a vaccine without one or more of the N-terminal and/orC-terminal modifications that are particularly advantageous forexpression of such proteins in yeast. Such HCV fusion proteins areuseful in other non-yeast based vaccines, such as by combining thefusion proteins with a conventional adjuvant, pulsing dendritic cellswith such fusion proteins, providing DNA or nucleic acid or viral vectorvaccines including nucleic acid molecules encoding such fusion proteins,or constructing pseudovirions compose of particular HCV fusion proteinsof the invention (e.g., E1-E2 fusions of the invention).

Accordingly, yet another embodiment of the present invention relates toa composition to protect an animal against HCV infection or a symptomresulting from such infection, the composition (which can be a vaccine)comprising: (a) any one or more of the HCV fusion proteins as describedabove (with or without the various N- and C-terminal modificationsdescribed herein); and (b) a pharmaceutically acceptable deliveryvehicle (which can include a pharmaceutically acceptable excipient oradjuvant).

Yet another embodiment of the present invention relates to a nucleicacid-based vaccine, such as a DNA vaccine or viral vector vaccine,comprising a nucleic acid construct (e.g., a viral vector or otherrecombinant nucleic acid molecule) encoding an HCV fusion protein asdescribed herein (with or without the various N- and C-terminalmodifications described herein). The vaccine can further include anypharmaceutically acceptable delivery vehicle (which can include apharmaceutically acceptable excipient or adjuvant).

Another embodiment of the present invention relates to a pseudovirionwhich is composed of various HCV fusion proteins of the invention, andparticularly, an E1-E2 fusion as described herein. Again, the N- orC-terminal modifications that are particularly useful in connection witha yeast-based vaccine of the invention may be included or not included.

In one embodiment of the present invention, a composition or vaccine canalso include biological response modifier compounds, or the ability toproduce such modifiers (i.e., by transfection with nucleic acidmolecules encoding such modifiers), although such modifiers are notnecessary to achieve a robust immune response according to theinvention. For example, a yeast vehicle can be transfected with orloaded with at least one antigen and at least one biological responsemodifier compound, or a vaccine or composition of the invention can beadministered in conjunction with at least one biological responsemodifier. Biological response modifiers include compounds that canmodulate immune responses, which may be referred to as immunomodulatorycompounds. Certain biological response modifiers can stimulate aprotective immune response whereas others can suppress a harmful immuneresponse. Certain biological response modifiers preferentially enhance acell-mediated immune response whereas others preferentially enhance ahumoral immune response (i.e., can stimulate an immune response in whichthere is an increased level of cellular compared to humoral immunity, orvice versa.). There are a number of techniques known to those skilled inthe art to measure stimulation or suppression of immune responses, aswell as to differentiate cellular immune responses from humoral immuneresponses.

Suitable biological response modifiers include cytokines, hormones,lipidic derivatives, small molecule drugs and other growth modulators,such as, but not limited to, interleukin 2 (IL-2), interleukin 4 (IL-4),interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma(IFN-gamma) insulin-like growth factor I (IGF-I), transforming growthfactor beta (TGF-β) steroids, prostaglandins and leukotrienes. Theability of a yeast vehicle to express (i.e., produce), and possiblysecrete, IL-2, IL-12 and/or IFN-gamma preferentially enhancescell-mediated immunity, whereas the ability of a yeast vehicle toexpress, and possibly secrete, IL-4, IL-5 and/or IL-10 preferentiallyenhances humoral immunity. Other suitable biological response modifiersinclude, but are not limited to, anti-CTLA-4 antibody (e.g., to releaseanergic T cells); T cell co-stimulators (e.g., anti-CD137, anti-CD28,anti-CD40); alemtuzumab (e.g., CamPath®), denileukin diftitox (e.g.,ONTAK®), anti-CD4, anti-CD25, anti-PD-1, anti-PD-L1, anti-PD-L2 oragents that block FOXP3 (e.g., to abrogate the activity/kill CD4+/CD25+Tregulatory cells); F1t3 ligand, imiquimod (Aldara™), GM-CSF,sargramostim (Leukine®), Toll-like receptor (TLR)-7 agonists, or TLR-9agonists (e.g., agents that increase the number of, or increase theactivation state, of dendritic cells, macrophages and other professionalantigen-presenting cells). Such biological response modifiers are wellknown in the art and are publicly available.

Compositions and therapeutic vaccines of the invention can furtherinclude any other compounds that are useful for protecting a subjectfrom HCV infection or that treats or ameliorates any symptom of such aninfection.

As mentioned above, the present invention also includes the use of anyof the HCV fusion proteins described herein, or a nucleic acid moleculeencoding such HCV fusion proteins, in a composition or vaccine in theabsence of the yeast vehicle of the present invention, such as in anyconventional or non-yeast-based composition or vaccine. Such acomposition can include, in addition to the HCV fusion protein, apharmaceutically acceptable carrier, such as an adjuvant. In addition,yeast-based vaccines of the invention may be provided in conjunctionwith a pharmaceutically acceptable carrier.

As used herein, a pharmaceutically acceptable carrier refers to anysubstance or vehicle suitable for delivering an HCV fusion proteinuseful in a method of the present invention to a suitable in vivo or exvivo site. Such a carrier can include, but is not limited to, anadjuvant, an excipient, or any other type of delivery vehicle orcarrier.

According to the present invention, adjuvants are typically substancesthat generally enhance the immune response of an animal to a specificantigen. Suitable adjuvants include, but are not limited to, Freund'sadjuvant; other bacterial cell wall components; aluminum-based salts;calcium-based salts; silica; polynucleotides; toxoids; serum proteins;viral coat proteins; other bacterial-derived preparations; gammainterferon; block copolymer adjuvants, such as Hunter's Titermaxadjuvant (CytRx™, Inc. Norcross, Ga.); Ribi adjuvants (available fromRibi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and theirderivatives, such as Quil A (available from Superfos Biosector A/S,Denmark).

Carriers are typically compounds that increase the half-life of atherapeutic composition in the treated animal. Suitable carriersinclude, but are not limited to, polymeric controlled releaseformulations, biodegradable implants, liposomes, oils, esters, andglycols.

Therapeutic compositions of the present invention can also contain oneor more pharmaceutically acceptable excipients. As used herein, apharmaceutically acceptable excipient refers to any substance suitablefor delivering a therapeutic composition useful in the method of thepresent invention to a suitable in vivo or ex vivo site. Preferredpharmaceutically acceptable excipients are capable of maintaining acomposition (or a yeast vehicle or dendritic cell comprising the yeastvehicle) in a form that, upon arrival of the composition at a targetcell, tissue, or site in the body, the composition is capable ofeliciting an immune response at the target site (noting that the targetsite can be systemic). Suitable excipients of the present inventioninclude excipients or formularies that transport, but do notspecifically target the vaccine to a site (also referred to herein asnon-targeting carriers). Examples of pharmaceutically acceptableexcipients include, but are not limited to water, saline, phosphatebuffered saline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity. Suitable auxiliary substances include, forexample, sodium acetate, sodium chloride, sodium lactate, potassiumchloride, calcium chloride, and other substances used to producephosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliarysubstances can also include preservatives, such as thimerosal, m- oro-cresol, formalin and benzol alcohol.

Methods of the Invention

Another embodiment of the present invention relates to a method toprotect an animal against an HCV infection or disease resultingtherefrom. The method includes the step of administering to an animalthat has or is at risk of developing a HCV infection, a vaccine orcomposition of the present invention as described herein, to reduce orprevent the HCV infection or at least one symptom resulting from the HCVinfection in the animal.

Yet another embodiment of the present invention relates to a method toelicit an antigen-specific humoral immune response and/or anantigen-specific cell-mediated immune response in an animal. The methodincludes administering to the animal a vaccine or composition of thepresent invention as described herein. The method of the presentinvention preferentially elicits an antigen-specific cell-mediatedimmune response in an animal.

In the above-embodiments, the vaccine or composition can include (1) acomposition comprising (a) a yeast vehicle; and (b) any one or more ofthe above-described HCV fusion proteins; and/or (2) (a) any one or moreof the above-described HCV fusion proteins; and (b) a pharmaceuticallyacceptable delivery vehicle (which can include or consist of apharmaceutically acceptable excipient or adjuvant); and/or (3) (a) anisolated nucleic acid molecule (e.g., a DNA construct, a vector, a viralvector) encoding any one or more of the above-described HCV fusionproteins; and/or (4) isolated dendritic cells (e.g., autologousdendritic cells containing (pulsed with) (a) a yeast vehicle; and/or (b)any one or more of the above-described HCV fusion proteins; and/or (5)HCV pseudovirions composed of any of the E1-E2 containing HCV fusionproteins of described herein.

In one embodiment of the present invention, the vaccine or compositionof the invention as described herein can be administered in a protocolthat includes the administration of one or more other vaccine orimmunotherapy compositions, including any conventional vaccine orcomposition. For example, such other vaccines or immunotherapycompositions can include any other antigen-containing, antigen-encoding,or antigen-expressing composition, such as a DNA vaccine encoding an HCVantigen or other viral vectors comprising an HCV antigen. Viral vectorsfor vaccines are known in the art and include, but are not limited to,pox viruses (vaccinia, canary, avipox), adeno viruses, adeno-associatedviruses, alpha viruses (Sindbis, VEE). Other types of vaccines,including protein-based vaccines, are also encompassed by thisembodiment. In one aspect, such a conventional vaccine or vaccine thatis not a part of the present invention or a vaccine of the presentinvention that does not include a yeast vehicle (e.g., a vaccinecomprising a novel HCV fusion protein of the invention in combinationwith a pharmaceutically acceptable carrier, or a DNA vaccine encoding anovel HCV fusion protein of the invention) can be administered initiallyto a subject to prime the immune response of the subject against the HCVantigen(s). Subsequently, the vaccine or composition of the presentinvention, and particularly, a yeast-based vaccine of the presentinvention, can be administered to the subject in order to boost theimmune response. Alternatively, the vaccine or composition of thepresent invention can be administered to the subject to prime the immuneresponse, including particularly a yeast-based vaccine of the presentinvention, and the conventional or other vaccine or composition (e.g., anon-yeast-based vaccine comprising a novel HCV fusion protein of theinvention or DNA vaccine encoding a novel HCV fusion protein of theinvention) can be used to boost the response.

The method of use of the therapeutic composition or vaccine of thepresent invention preferably elicits an immune response in an animalsuch that the animal is protected from HCV infection or from diseaseconditions or symptoms resulting from HCV infection. As used herein, thephrase “protected from a disease” refers to reducing the symptoms of thedisease; reducing the occurrence of the disease, and/or reducing theseverity of the disease. Protecting an animal can refer to the abilityof a therapeutic composition of the present invention, when administeredto an animal, to prevent a disease from occurring and/or to cure or toalleviate disease symptoms, signs or causes. As such, to protect ananimal from a disease includes both preventing disease occurrence(prophylactic treatment or prophylactic vaccine) and treating an animalthat has a disease or that is experiencing initial symptoms of a disease(therapeutic treatment or a therapeutic vaccine). In particular,protecting an animal from a disease is accomplished by eliciting animmune response in the animal by inducing a beneficial or protectiveimmune response which may, in some instances, additionally suppress(e.g., reduce, inhibit or block) an overactive or harmful immuneresponse. The term, “disease” refers to any deviation from the normalhealth of an animal and includes a state when disease symptoms arepresent, as well as conditions in which a deviation (e.g., infection,gene mutation, genetic defect, etc.) has occurred, but symptoms are notyet manifested.

In one embodiment, any of the vaccines of the present invention isadministered to an individual, or to a population of individuals, whohave been infected with HCV. In another embodiment, any of the vaccinesof the present invention is administered to an individual, or to apopulation of individuals, who are at risk of being infected with HCV.Such individuals can include populations identified as higher-risk forHCV infection than, for example, the normal or entire population ofindividuals. Such populations can be defined by any suitable parameter.In another embodiment, any of the vaccines of the present invention isadministered to any individual, or to any population of individuals,regardless of their known or predicted infection status orsusceptibility to becoming infected with HCV.

More specifically, a vaccine as described herein, when administered toan animal by the method of the present invention, preferably produces aresult which can include alleviation of the disease (e.g., reduction ofat least one symptom or clinical manifestation of the disease),elimination of the disease, prevention or alleviation of a secondarydisease resulting from the occurrence of a primary disease, preventionof the disease, and stimulation of effector cell immunity against thedisease.

The present invention includes the delivery of a composition or vaccineof the invention to an animal. The administration process can beperformed ex vivo or in vivo. Ex vivo administration refers toperforming part of the regulatory step outside of the patient, such asadministering a composition of the present invention to a population ofcells (dendritic cells) removed from a patient under conditions suchthat a yeast vehicle and antigen are loaded into the cell, and returningthe cells to the patient. The therapeutic composition of the presentinvention can be returned to a patient, or administered to a patient, byany suitable mode of administration.

Administration of a vaccine or composition, including a dendritic cellloaded with the yeast vehicle and antigen, a yeast vehicle alone, or acomposition comprising a novel HCV fusion protein, alone or incombination with a carrier according to the present invention, can besystemic, mucosal and/or proximal to the location of the target site(e.g., near a tumor). The preferred routes of administration will beapparent to those of skill in the art, depending on the type ofcondition to be prevented or treated, the antigen used, and/or thetarget cell population or tissue. Preferred methods of administrationinclude, but are not limited to, intravenous administration,intraperitoneal administration, intramuscular administration, intranodaladministration, intracoronary administration, intraarterialadministration (e.g., into a carotid artery), subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, aural, intranasal, oral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue. Particularly preferred routes of administrationinclude: intravenous, intraperitoneal, subcutaneous, intradermal,intranodal, intramuscular, transdermal, inhaled, intranasal, oral,intraocular, intraarticular, intracranial, and intraspinal. Parenteraldelivery can include intradermal, intramuscular, intraperitoneal,intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheterand venal catheter routes. Aural delivery can include ear drops,intranasal delivery can include nose drops or intranasal injection, andintraocular delivery can include eye drops. Aerosol (inhalation)delivery can also be performed using methods standard in the art (see,for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference in itsentirety). For example, in one embodiment, a composition or vaccine ofthe invention can be formulated into a composition suitable fornebulized delivery using a suitable inhalation device or nebulizer. Oraldelivery can include solids and liquids that can be taken through themouth, and is useful in the development of mucosal immunity and sincecompositions comprising yeast vehicles can be easily prepared for oraldelivery, for example, as tablets or capsules, as well as beingformulated into food and beverage products. Other routes ofadministration that modulate mucosal immunity are useful in thetreatment of viral infections. Such routes include bronchial,intradermal, intramuscular, intranasal, other inhalatory, rectal,subcutaneous, topical, transdermal, vaginal and urethral routes.

In one embodiment of any of the above-identified methods, the vaccine isadministered to the respiratory tract. In another embodiment, thevaccine is administered by a parenteral route of administration. In yetanother embodiment, the vaccine further comprises dendritic cells ormacrophages, wherein a yeast vehicle expressing the fusion protein isdelivered to dendritic cells or macrophages ex vivo and wherein thedendritic cell or macrophage containing the yeast vehicle expressing theHCV antigen is administered to the animal. In one aspect of thisembodiment, the dendritic cell or the yeast vehicle has beenadditionally loaded with free antigen. In one aspect, the vaccine isadministered as a therapeutic vaccine. In another aspect, the vaccine isadministered as a prophylactic vaccine.

According to the present invention, an effective administration protocol(i.e., administering a vaccine or therapeutic composition in aneffective manner) comprises suitable dose parameters and modes ofadministration that result in elicitation of an immune response in ananimal that has a disease or condition, or that is at risk ofcontracting a disease or condition, preferably so that the animal isprotected from the disease. Effective dose parameters can be determinedusing methods standard in the art for a particular disease. Such methodsinclude, for example, determination of survival rates, side effects(i.e., toxicity) and progression or regression of disease.

In accordance with the present invention, a suitable single dose size isa dose that is capable of eliciting an antigen-specific immune responsein an animal when administered one or more times over a suitable timeperiod. Doses can vary depending upon the disease or condition beingtreated. For example, in one embodiment, a single dose of a yeastvehicle of the present invention is from about 1×10⁵ to about 5×10⁷yeast cell equivalents per kilogram body weight of the organism beingadministered the composition. In a preferred embodiment, the yeast cellsper dose are not adjusted for weight of the organism. In thisembodiment, a single dose of a yeast vehicle of the present invention isfrom about 1×10⁴ to about 1×10⁹ yeast cells per dose. More preferably, asingle dose of a yeast vehicle of the present invention is from about0.1 Y.U. (1×10⁶ cells) to about 100 Y.U. (1×10⁹ cells) per dose (i.e.,per organism), including any interim dose, in increments of 0.1×10⁶cells (i.e., 1.1×10⁶, 1.2×10⁶, 1.3×10⁶ . . . ). This range of doses canbe effectively used in any organism of any size, including mice,monkeys, humans, etc.

When the vaccine is administered by loading the yeast vehicle andantigen into dendritic cells, a preferred single dose of a vaccine ofthe present invention is from about 0.5×10⁶ to about 40×10⁶ dendriticcells per individual per administration. Preferably, a single dose isfrom about 1×10⁶ to about 20×10⁶ dendritic cells per individual, andmore preferably from about 1×10⁶ to about 10×10⁶ dendritic cells perindividual.

When the vaccine comprises a fusion protein of the present invention anda carrier, a preferred single dose is from about 0.01microgram×kilogram⁻¹ and about 10 milligram×kilogram⁻¹ body weight of ananimal. A more preferred single dose of an agent comprises between about1 microgram×kilogram⁻¹ and about 10 milligram×kilogram⁻¹ body weight ofan animal. An even more preferred single dose of an agent comprisesbetween about 5 microgram×kilogram⁻¹ and about 7 milligram×kilogram⁻¹body weight of an animal. An even more preferred single dose of an agentcomprises between about 10 microgram×kilogram⁻¹ and about 5milligram×kilogram⁻¹ body weight of an animal. A particularly preferredsingle dose of an agent comprises between about 0.1 milligram×kilogram⁻¹and about 5 milligram×kilogram⁻¹ body weight of an animal, if the anagent is delivered by aerosol. Another particularly preferred singledose of an agent comprises between about 0.1 microgram×kilogram⁻¹ andabout 10 microgram×kilogram⁻¹ body weight of an animal, if the agent isdelivered parenterally.

“Boosters” or “boosts” of a therapeutic composition are preferablyadministered when the immune response against the antigen has waned oras needed to provide an immune response or induce a memory responseagainst a particular antigen or antigen(s). Boosters can be administeredfrom about 2 weeks to several years after the original administration.In one embodiment, an administration schedule is one in which from about1×10⁵ to about 5×10⁷ yeast cell equivalents of a composition per kg bodyweight of the organism is administered from about one to about 4 timesover a time period of from about 1 month to about 6 months.

In the method of the present invention, vaccines and therapeuticcompositions can be administered to animal, including any vertebrate,and particularly to any member of the Vertebrate class, Mammalia,including, without limitation, primates, rodents, livestock and domesticpets. Livestock include mammals to be consumed or that produce usefulproducts (e.g., sheep for wool production). Preferred mammals to protectinclude humans, dogs, cats, mice, rats, goats, sheep, cattle, horses andpigs, with humans being particularly preferred. According to the presentinvention, the term “patient” or “subject” can be used to describe anyanimal that is the subject of a diagnostic, prophylactic, or therapeutictreatment as described herein.

Isolated Fusion Proteins, Nucleic Acid Molecules, and Cells

Another embodiment of the present invention includes an isolatedprotein, comprising any of the isolated fusion protein comprising an HCVantigen(s) as described herein. Also included in the present inventionare isolated nucleic acid molecules encoding any of such proteins,recombinant nucleic acid molecules comprising nucleic acid sequencesencoding such proteins, and cells and vectors, including viral vectors,that contain or are transfected/transformed with such nucleic acidmolecules or recombinant nucleic acid molecules.

As used herein, reference to an isolated protein or polypeptide in thepresent invention includes full-length proteins, fusion proteins, or anyfragment, domain, conformational epitope, or homologue of such proteins.More specifically, an isolated protein, according to the presentinvention, is a protein (including a polypeptide or peptide) that hasbeen removed from its natural milieu (i.e., that has been subject tohuman manipulation) and can include purified proteins, partiallypurified proteins, recombinantly produced proteins, and syntheticallyproduced proteins, for example. As such, “isolated” does not reflect theextent to which the protein has been purified. Preferably, an isolatedprotein of the present invention is produced recombinantly. According tothe present invention, the terms “modification” and “mutation” can beused interchangeably, particularly with regard to themodifications/mutations to the amino acid sequence of proteins orportions thereof (or nucleic acid sequences) described herein.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by minor modifications tothe naturally occurring protein or peptide, but which maintains thebasic protein and side chain structure of the naturally occurring form.Such changes include, but are not limited to: changes in one or a fewamino acid side chains; changes one or a few amino acids, includingdeletions (e.g., a truncated version of the protein or peptide)insertions and/or substitutions; changes in stereochemistry of one or afew atoms; and/or minor derivatizations, including but not limited to:methylation, glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue can have either enhanced,decreased, or substantially similar properties as compared to thenaturally occurring protein or peptide. A homologue can include anagonist of a protein or an antagonist of a protein. Homologues can beproduced using techniques known in the art for the production ofproteins including, but not limited to, direct modifications to theisolated, naturally occurring protein, direct protein synthesis, ormodifications to the nucleic acid sequence encoding the protein using,for example, classic or recombinant DNA techniques to effect random ortargeted mutagenesis.

The minimum size of a protein and/or a homologue or fragment or otherportion thereof of the present invention is, in one aspect, a sizesufficient to have the requisite biological activity, such as serving asan antigen(s) or immunogen(s) in a fusion protein or other compositionof the invention, or as a target in an in vitro assay. In oneembodiment, a protein of the present invention is at least about 8 aminoacids in length, or at least about 25 amino acids in length, or at leastabout 30 amino acids in length, or at least about 40 amino acids inlength, or at least about 50 amino acids in length, or at least about 75amino acids in length, or at least about 100 amino acids in length, orat least about 125 amino acids in length, or at least about 150 aminoacids in length, or at least about 175 amino acids in length, or atleast about 200 amino acids in length, or at least about 250 amino acidsin length, or at least about 300 amino acids in length, or at leastabout 350 amino acids in length, or at least about 400 amino acids inlength, or at least about 450 amino acids in length, or at least about500 amino acids in length, or at least about 550 amino acids in length,or at least about 600 amino acids in length, and so on, in any lengthbetween 8 amino acids and up to the full length of a protein of theinvention, the full-length of a combination of proteins or portionsthereof, or longer, in whole integers (e.g., 8, 9, 10, . . . 25, 26, . .. 102, 103, . . . ). There is no limit, other than a practical limit, onthe maximum size of such a protein in that the protein can include aportion of a protein, a functional domain, or a biologically active oruseful fragment thereof, or a full-length protein, plus additionalsequence (e.g., a fusion protein sequence), if desired.

Preferred fusion proteins according to the present invention include anyof the fusion proteins described herein. Exemplary fusion proteinsencompassed by the present invention include those fusion proteinscomprising, consisting essentially of, or consisting of, and amino acidsequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 AND SEQ ID NO:18. Otherfusion protein sequences will be apparent to those of skill in the artgiven the guidance provided herein, since various HCV protein sequencesare well-known in the art.

The present invention also includes any nucleic acid moleculescomprising, consisting essentially of, or consisting of, a nucleic acidsequence encoding any of the fusion proteins described herein. Inaccordance with the present invention, an isolated nucleic acid moleculeis a nucleic acid molecule that has been removed from its natural milieu(i.e., that has been subject to human manipulation), its natural milieubeing the genome or chromosome in which the nucleic acid molecule isfound in nature. As such, “isolated” does not necessarily reflect theextent to which the nucleic acid molecule has been purified, butindicates that the molecule does not include an entire genome or anentire chromosome in which the nucleic acid molecule is found in nature.An isolated nucleic acid molecule can include a gene. An isolatednucleic acid molecule that includes a gene is not a fragment of achromosome that includes such gene, but rather includes the codingregion and regulatory regions associated with the gene, but noadditional genes that are naturally found on the same chromosome. Anisolated nucleic acid molecule can also include a specified nucleic acidsequence flanked by (i.e., at the 5′ and/or the 3′ end of the sequence)additional nucleic acids that do not normally flank the specifiednucleic acid sequence in nature (i.e., heterologous sequences). Isolatednucleic acid molecule can include DNA, RNA (e.g., mRNA), or derivativesof either DNA or RNA (e.g., cDNA). Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a protein or domainof a protein.

Preferably, an isolated nucleic acid molecule of the present inventionis produced using recombinant DNA technology (e.g., polymerase chainreaction (PCR) amplification, cloning) or chemical synthesis. Isolatednucleic acid molecules include natural nucleic acid molecules andhomologues thereof, including, but not limited to, natural allelicvariants and modified nucleic acid molecules in which nucleotides havebeen inserted, deleted, substituted, and/or inverted in such a mannerthat such modifications provide the desired effect. Protein homologues(e.g., proteins encoded by nucleic acid homologues) have been discussedin detail above.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress (1989)). For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, PCR amplification and/ormutagenesis of selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

A recombinant nucleic acid molecule expressing a fusion protein of thepresent invention is a molecule that can include at least one of anynucleic acid sequence encoding any one or more fusion proteins describedherein operatively linked to at least one of any transcription controlsequence capable of effectively regulating expression of the nucleicacid molecule(s) in the cell to be transfected. Although the phrase“nucleic acid molecule” primarily refers to the physical nucleic acidmolecule and the phrase “nucleic acid sequence” primarily refers to thesequence of nucleotides on the nucleic acid molecule, the two phrasescan be used interchangeably, especially with respect to a nucleic acidmolecule, or a nucleic acid sequence, being capable of encoding aprotein. In addition, the phrase “recombinant molecule” primarily refersto a nucleic acid molecule operatively linked to a transcription controlsequence, but can be used interchangeably with the phrase “nucleic acidmolecule” which is administered to an animal.

A recombinant nucleic acid molecule includes a recombinant vector, whichis any nucleic acid sequence, typically a heterologous sequence, whichis operatively linked to the isolated nucleic acid molecule encoding afusion protein of the present invention, which is capable of enablingrecombinant production of the fusion protein, and which is capable ofdelivering the nucleic acid molecule into a host cell according to thepresent invention. Such a vector can contain nucleic acid sequences thatare not naturally found adjacent to the isolated nucleic acid moleculesto be inserted into the vector. The vector can be either RNA or DNA,either prokaryotic or eukaryotic, and preferably in the presentinvention, is a virus or a plasmid. Recombinant vectors can be used inthe cloning, sequencing, and/or otherwise manipulating of nucleic acidmolecules, and can be used in delivery of such molecules (e.g., as in aDNA vaccine or a viral vector-based vaccine). Recombinant vectors arepreferably used in the expression of nucleic acid molecules, and canalso be referred to as expression vectors. Preferred recombinant vectorsare capable of being expressed in a transfected host cell.

In a recombinant molecule of the present invention, nucleic acidmolecules are operatively linked to expression vectors containingregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the host cell and thatcontrol the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude nucleic acid molecules that are operatively linked to one ormore transcription control sequences. The phrase “operatively linked”refers to linking a nucleic acid molecule to a transcription controlsequence in a manner such that the molecule is expressed whentransfected (i.e., transformed, transduced or transfected) into a hostcell.

Transcription control sequences are sequences that control theinitiation, elongation, and termination of transcription. Particularlyimportant transcription control sequences are those that controltranscription initiation, such as promoter, enhancer, operator andrepressor sequences. Suitable transcription control sequences includeany transcription control sequence that can function in a host cellaccording to the present invention. A variety of suitable transcriptioncontrol sequences are known to those skilled in the art.

According to the present invention, the term “transfection” is used torefer to any method by which an exogenous nucleic acid molecule (i.e., arecombinant nucleic acid molecule) can be inserted into a cell. The term“transformation” can be used interchangeably with the term“transfection” when such term is used to refer to the introduction ofnucleic acid molecules into microbial cells, such as algae, bacteria andyeast, or into plant cells. In microbial systems and plant systems, theterm “transformation” is used to describe an inherited change due to theacquisition of exogenous nucleic acids by the microorganism or plant andis essentially synonymous with the term “transfection.” Therefore,transfection techniques include, but are not limited to, transformation,chemical treatment of cells, particle bombardment, electroporation,microinjection, lipofection, adsorption, infection and protoplastfusion.

One type of recombinant vector useful in a recombinant nucleic acidmolecule of the present invention is a recombinant viral vector. Such avector includes a recombinant nucleic acid sequence encoding a fusionprotein of the present invention that is packaged in a viral coat thatcan be expressed in a host cell in an animal or ex vivo afteradministration. A number of recombinant viral vectors can be used,including, but not limited to, those based on alphaviruses, poxviruses,adenoviruses, herpesviruses, lentiviruses, adeno-associated viruses andretroviruses. Particularly preferred viral vectors are those based onadenoviruses and adeno-associated viruses. Viral vectors suitable forgene delivery are well known in the art and can be selected by theskilled artisan for use in the present invention. A detailed discussionof current viral vectors is provided in “Molecular Biotechnology,”Second Edition, by Glick and Pasternak, ASM Press, Washington D.C.,1998, pp. 555-590, the entirety of which is incorporated herein byreference.

Suitable host cells to transfect with a recombinant nucleic acidmolecule according to the present invention include any cell that can betransfected or transformed, including any animal, insect, bacterial,fungal (including yeast) cell. In one embodiment, the host cell is ananimal cell, including a tumor cell, that has been transfected with andexpresses a fusion protein of the present invention. Such a cell isexemplified in the Examples section and is useful, for example, forassessing antigen-specific T cell responses that are induced by avaccine or composition of the present invention. Other vaccines orcompositions directed against an HCV antigen can also be tested suchtransfected tumor cells.

The following experimental results are provided for purposes ofillustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following example describes the engineering of GI-5005, a truncatedNS3-Core fusion protein yeast vaccine of the present invention.

The GI-5005 Saccharomyces cerevisiae was engineered to express a HCVNS3-Core fusion protein under the control of the copper-induciblepromoter, CUP1. Two regions of the HCV genome (genotype 1a, H77 strain,cDNA was provided by the NIH) were amplified by PCR in order to generatethe product. The NS3-Core fusion protein is a single polypeptide withthe following sequence elements fused in frame from N- to C-terminus(HCV polyprotein numbering in parentheses) (represented herein by SEQ IDNO:2): 1) the sequence MADEAP to impart resistance to proteasomaldegradation; 2) amino acids 89 to 350 (1115 to 1376) of the HCV NS3protease protein; 3) a single threonine amino acid residue introduced incloning; 4) amino acids 2 to 140 (2 to 140) of the HCV Core protein; and5) the sequence ED to increase the hydrophilicity of the Core variant.

Expression of the HCV NS3-Core fusion protein was confirmed by Westernblot analysis of lysates from copper-induced, heat-inactivated GI-5005yeast. Monoclonal antibodies specific for HCV NS3 (Virostat) or HCV Coreprotein (Anogen) were used for protein detection (See FIG. 1A and FIG.1B).

Example 2

The following example describes the engineering of GI-5003, aninactivated HCV NS3 yeast vaccine of the present invention.

The GI-5003 Saccharomyces cerevisiae was engineered to express aninactivated full-length HCV NS3 protein under the control of thecopper-inducible promoter, CUP1. A single region of the HCV genome(genotype 1a, H77 strain, cDNA was provided by the NIH) was amplified byPCR in order to generate the product. The inactivated NS3 protein is asingle polypeptide with the following sequence elements fused in framefrom N- to C-terminus (HCV polyprotein numbering in parentheses)(represented herein by SEQ ID NO:4): 1) the sequence MADEAP to impartresistance to proteasomal degradation; and 2) amino acids 1 to 631 (1027to 1657) of the HCV NS3 protease protein (note that the amino acid atHCV polypeptide residue 1165 has been changed from a serine to analanine in order to inactivate the proteolytic activity).

Expression of the HCV NS3 protein was confirmed by Western blot analysisof lysates from copper-induced, heat-inactivated GI-5003 yeast.Monoclonal antibodies specific for HCV NS3 (Virostat) were used forprotein detection (See FIG. 1A and FIG. 1B).

Example 3

The following example describes the engineering of the GI-5000 seriestruncated HCV E1-E2 fusion protein yeast vaccine of the presentinvention.

The E1-E2 fusion protein is a single polypeptide with the followingsequence elements fused in frame from N- to C-terminus (HCV polyproteinnumbering in parentheses) (represented herein by SEQ ID NO:6): 1) Thesequence MADEAP to impart resistance to proteasomal degradation, 2)amino acids 1 to 156 (192 to 347) of HCV protein E1, 3) amino acids 1 to334 (384-717) of HCV protein E2. 36 C-terminal hydrophobic amino acidsof E1 and 29 C-terminal hydrophobic amino acids of E2 were omitted fromthe fusion protein to promote cytoplasmic accumulation in yeast.

Expression of the HCV E1/E2 fusion protein was confirmed by Western blotanalysis of lysates from copper-induced, heat-inactivated yeast (SeeFIG. 1C).

Example 4

The following example describes the engineering of the GI-5000 series TMdomain-deleted HCV NS4b fusion protein yeast vehicle of the presentinvention.

The NS4b protein is a single polypeptide with the following sequenceelements arranged in tandem, in frame, from N- to C-terminus(polyprotein numbering in parentheses) (represented herein by SEQ IDNO:8): 1) The sequence MADEAP to impart resistance to proteosomaldegradation, 2) amino acids 1 to 69 (1712 to 1780) of HCV protein NS4b,3) amino acids 177 to 261 (1888 to 1972) of HCV protein NS4b. A 107amino acid region corresponding to NS4b amino acids 70 to 176 (1781 to1887) that contains multiple membrane spanning domains was omitted topromote cytoplasmic accumulation in yeast.

Expression of the HCV NS4b fusion protein was confirmed by Western blotanalysis of lysates from copper-induced, heat-inactivated yeast (SeeFIG. 1D).

Example 5

The following example describes non-clinical pharmacology studies inmice using the GI-5005 yeast vehicles (also referred to herein asTarmogen™™) expressing HCV antigens: immunogenicity studies.

GI-5005 consists of S. cerevisiae yeast (W303 strain obtained from theATCC) that have been stably transduced with a yeast expression plasmidencoding a fusion protein of truncated HCV genotype 1a-derived NS3 andcore gene products under the control of the yeast copper-inducible(CUP1) promoter (SEQ ID NO:2), as described in Example 1. In thefollowing studies, C57BL/6 (H-2^(b)) and BALB/cBy (H-2^(d)) mice wereinjected subcutaneously with GI-5005 yeast. In vitro and in vivo assaysthat detect induction of antigen-specific lymphocytes by GI-5005 wereemployed, including lymphocyte proliferation, cell-mediatedcytotoxicity, cytokine secretion, and protection from tumor challenge.To support these studies, the following yeast strains, cell lines andrecombinant viruses have been generated and maintained:

-   -   GI-5003: HCV-NS3 protein-expressing yeast strain. GI-5003        expresses full-length NS3 in which the catalytic domain has been        inactivated by a single point mutation.    -   GI-5005-L: GI-5005 yeast strain expressing less than 50 ng        HCV-NS3-Core fusion protein per YU.    -   GI-5005-M: GI-5005 yeast strain expressing approximately 500 ng        HCV-NS3-Core fusion protein per YU    -   GI-5005-H: GI-5005 yeast strain expressing approximately 1400 ng        HCV-N53-Core fusion protein per YU    -   EL4-NS3: C57BL/6-derived EL4 lymphoma cells (H-2^(b)) stably        transfected with DNA encoding HCV NS3.    -   A20-NS3: BALB/c-derived A20 lymphoma cells (H-2^(d)) stably        transfected with DNA encoding HCV NS3.    -   P815-NS3: DBA/2-derived P815 leukemia cells (H-2^(d)) stably        transfected with DNA encoding HCV NS3.    -   Recombinant vaccinia viruses (rVV) encoding beta-galactosidase        (rVV-lac), HIV-1 Gag (rVV-Gag), HCV NS3 (rVV-NS3) and HCV Core        (rVV-Core) proteins.

In the studies that are described below, and unless otherwise indicatedin a particular experiment, female BALB/c and/or C57BL/6 mice (5 pergroup; aged 6-10 weeks) were injected subcutaneously on a weekly basiswith 5 YU (50 million) GI-5005 or GI-5003 and were sacrificed seven daysafter the final injection. Spleen cell suspensions, pooled from eachgroup, were prepared in RPMI-1640 tissue culture medium supplementedwith 10% heat-inactivated fetal calf serum, L-glutamine, HEPES and2-mercaptoethanol and were subjected to in vitro stimulation (IVS)conditions utilizing both HCV antigen-specific (typically rVV-NS3 and/orrVV-Core) and yeast antigen-specific (typically GI-5005) stimuli asspecified. Standard assays were employed to evaluate immune responsesinduced by administration of GI-5005 and included lymphocyteproliferation as assessed by ³H-thymidine incorporation, cell-mediatedcytotoxicity assays employing ⁵¹Cr-labeled target cells, quantificationof cytokine secretion, and protection from tumor challenge.

(a) GI-5005 Induces Antigen-Specific Lymphocyte Proliferation.

In a preliminary experiment to evaluate the immunogenicity of GI-5005,C57BL/6 mice were injected weekly for three weeks with 5 YU (50 million)heat-inactivated GI-5005 yeast cells. The mice showed no apparentadverse effects from immunization. Spleen cells were obtained seven daysafter the final immunization and single cell suspensions were stimulatedin vitro with either nothing, EL4 lymphoma cells, EL4-NS3 (EL4 stablyexpressing HCV NS3), rVV-NS3 (recombinant vaccinia virus encoding HCVNS3) or rVV-Core. Lymphocyte proliferation was assessed using a standardthymidine incorporation assay after 5 days in culture. Morespecifically, spleen cells from C57BL/6 mice that were injected with 5YU GI-5005 were placed in individual wells of 96-well U-bottomed tissueculture plates (400,000 cells/well) and stimulated in vitro with:nothing, mitomycin C-treated EL4 (10,000 cells/well), mitomycinC-treated EL4-NS3 (10,000 cells/well), rVV-NS3 (400,000 pfu/well) orrVV-Core (400,000 pfu/well). 3HTdR was added on day 5 and the plateswere harvested 18 hours thereafter. Results are expressed as the averageCPM+/−S.D. for triplicate samples. The results presented in FIG. 2 showthat GI-5005 induces NS3- and Core-specific lymphocyte proliferation.

(b) GI-5005 Induces Antigen-Specific Cytotoxic Effector Cell Responses:

GI-5005 Induces Cytotoxic Effector Cells that Kill Tumor Cells StablyExpressing HCV NS3

FIG. 3 shows that immunization with GI-5005 induces cytotoxic effectorcells that can kill HCV NS3-expressing tumor cells. Specifically, spleencells from C57BL/6 mice that were injected weekly for three weeks with 5YU GI-5005 were placed in individual wells of either 25 cm² tissueculture flasks at 30×10⁶ cells/flask (FIGS. 3A-3B) or 24-wellflat-bottomed tissue culture plates at 6×10⁶/well (FIG. 3C). Spleencells were stimulated in vitro (IVS) for 6 days with either 1 YU (10⁷yeast cells) GI-5005/flask (FIG. 3A); with nothing, 1 YU/flask GI-1001or 1 YU/flask GI-5005 (FIG. 3B); or with 6×10⁶ pfu/well rVV-lac orrVV-NS3 (FIG. 3C). Spleen cells in culture with rVV-lac or rVV-NS3 wereexpanded for an additional 3 days in the presence of 10% T-stim as asource of T cell growth factors. At the end of the 6 (FIGS. 3A,3B) or 9(FIG. 3C) day IVS culture period, doubling dilutions of the spleen cellcultures were mixed with ten thousand ⁵¹Cr-labeled EL4 or EL4-NS3 cellsas indicated. E:T ratio refers to the effector:target ratio based onspleen effector cell concentrations at the start of the IVS cultureperiod. Results are expressed as the average percent specific lysis+/−S.D. for triplicate samples isolated after six hours of co-culture in96-well V-bottomed plates. Percent spontaneous chromium release valueswere 19% for EL-4 (FIG. 3A), 40% for EL4-NS3 (FIGS. 3A,3B) and 29% forEL4-NS3 (FIG. 3C).

In the results presented in FIG. 3A, spleen cells derived from GI-5005immunized C57BL/6 mice were stimulated in vitro with GI-5005 yeast, at ayeast to spleen cell ratio of 1:3, for 6 days prior to testing on⁵¹Cr-labeled non-transfected EL4 lymphoma cells or on EL4 cells stablyexpressing HCV-NS3 (EL4-N53). The stimulated spleen cells killed EL4-NS3targets in a dose-dependent manner. In contrast, significantly lesskilling was observed on non-transfected EL4 cells. In addition toproviding evidence that immunization with GI-5005 induced NS3-specificcytotoxic effector cells, these data also indicated that GI-5005 yeastcould be used in vitro to re-stimulate NS3-specific cytotoxic effectorcells. The results presented in FIG. 3B provide further confirmation forthis finding and show that in vitro stimulation (IVS) of spleen cellsfrom GI-5005-immunized mice with GI-5005 reveals cytotoxic effectorcells capable of enhanced cytotoxic activity against EL4-N53, ascompared to IVS with nothing (nil) or with vector control yeast(GI-1001). The requirement for exposure to some form of HCV NS3 antigento activate cytotoxic effector cells activity during the IVS period wasfurther investigated using stimulation with recombinant vaccinia virusencoding NS3 (rVV-NS3; FIG. 3C). These data show that NS3-specificcytotoxic effector cells present in the spleen of immunized mice arestimulated by IVS with rVV-NS3 as compared to IVS with rVV-lac, arecombinant virus encoding the irrelevant antigen beta-galactosidase.

GI-5005 Induces Cytotoxic Effector Cells that Kill Tumor Cells Infectedwith Recombinant Vaccinia Virus Encoding HCV NS3 or Core

The results presented above demonstrated that immunization with GI-5005leads to induction of cytotoxic effector cells that can kill syngeneictumor cells expressing NS3. However, GI-5005 also expresses the HCV Coreantigen. Attempts to derive stably transfected tumor cell linesexpressing HCV Core protein were unsuccessful. To overcome the lack of aCore-expressing target cell, the studies presented in FIG. 4 wereperformed. In brief, H-2^(d)-bearing P815 leukemia cells were infectedovernight with recombinant vaccinia viruses encoding either HCV NS3 orHCV Core prior to their use in a standard chromium release assayemploying spleen cells from BALB/c mice that had been immunized witheither GI-5005 or GI-5003 (a Tarmogen™™ expressing full-length HCV-NS3but not Core) and stimulated in vitro for 5 days in the presence ofGI-5005. More particularly, spleen cells from BALB/c mice that wereinjected weekly for three weeks with 5 YU GI-5005 (GI-5005) or 5 YUGI-5003 (GI-5003) were placed in individual wells of 24-wellflat-bottomed tissue culture plates (8×10⁶/well) and were stimulated invitro with GI-5005 (1×10⁶/well) for 5 days. At the end of the IVSculture period, doubling dilutions of the spleen cell cultures weremixed with ten thousand 51Cr-labeled P815 leukemia cells that had beeninfected overnight with recombinant vaccinia virus encoding HCV NS3(FIG. 4A) or HCV Core (FIG. 4B). E:T ratio refers to the effector:targetcell ratio based on spleen effector cell concentrations at the start ofthe IVS culture period. Results are expressed as the average percentspecific lysis +/−S.D. for triplicate samples isolated after 6 hours ofco-culture in 96-well V-bottomed plates. Percent spontaneous chromiumrelease values were 21% for P815-rVV-NS3 and 40% for P815-rVV-Core.

FIGS. 4A and 4B shows that GI-5005 induces cytotoxic cells that can killtumor cells infected with either rVV-NS3 (FIG. 4A) or rVV-Core (FIG. 4B)whereas killing induced by GI-5003 is restricted to NS3. In summary, theresults presented in FIGS. 3 and 4 indicate that immunization withGI-5005 induces NS3- and Core-specific cytotoxic effector cell activity.

(c) GI-5005 Induces Cells that Secrete Pro-Inflammatory Cytokines

FIG. 5 shows the cytokines that are secreted when spleen cells fromeither naïve or GI-5005 immunized C57BL/6 mice are placed in tissueculture with GI-5005 yeast. Cell-free supernatants were collected 48hours after initiation of culture and cytokine concentrations weredetermined using the flow cytometer-based Luminex™ assay (Biosource).More specifically, spleen cells from naïve C57BL/6 mice or from C57BL/6mice that received three weekly injections of 5 YU GI-5005 were placedin individual wells of 24-well flat-bottomed tissue culture plates(10×10⁶/well). Spleen cells were stimulated with either GI-5005 (1×10⁶yeast cells/well) or PMA (15 ng/mL) plus Ionomycin (750 ng/mL).Cell-free supernatants were collected at 48 hours after initiation ofculture and cytokines were quantified by the University of ColoradoCancer Center Flow Cytometer Facility using the flow-cytometer Luminex™assay (Biosource). IFN-g=IFN-γ; TNF-a=TNF-α.

These results show that GI-5005 administration elicits T cells thatsecrete IL-2 and IL-5, as well as the pro-inflammatory cytokines IL-6,GM-CSF, IFN-γ and TNF-α. It is important to note that the cytokineresponse of spleen cells from immunized mice exposed to yeast in vitrois comparable in magnitude to that observed upon polyclonal stimulationof T cells from naïve C57BL/6 mice with PMA plus ionomycin. In addition,FIG. 5 also shows the cytokine response of naïve C57BL/6 spleen cells toyeast and indicates that the innate response to yeast includes secretionof IL-6, IL-12 and TNF-α, presumably derived from monocytes anddendritic cells in the population. Similar results were obtained withspleen cells from naïve and immunized BALB/c mice (see FIGS. 8 and 11).

(d) Effect of Repeated Administration on Immune Responses Induced withGI-5005

The results presented in FIGS. 6, 7 and 8 are from experiments comparingone, two or three weekly immunizations with GI-5005 conducted in bothC57BL/6 and BALB/c mice. FIG. 6 examines NS3- and Core-specificlymphocyte proliferation, FIG. 7 shows the induction of NS3- andCore-specific cytotoxic cell activity and FIG. 8 shows cytokinesecretion profiles. Overall, these results indicate that a singleinjection of GI-5005 induces a weak response that is significantlyenhanced by additional administrations.

FIG. 6 shows the results of a lymphocyte proliferation assay performedwith spleen cells from C57BL/6 mice that received one, two or threeweekly immunizations with GI-5005. Specifically, spleen cells fromC57BL/6 mice that received one, two or three weekly injections with 5YUGI-5005 were placed in individual wells of 96-well U-bottomed tissueculture plates (400,000 cells/well) and stimulated in vitro with eithernothing, rVV-N53, rVV-Core or rVV-rastafar (100,000 pfu/well). 3HTdR wasadded on day 5 and the plates were harvested 18 hours thereafter.Results are expressed as the average CPM +/−S.D. for triplicate samples.The response of HCV NS3 and Core-specific lymphocytes increased inproportion with the number of immunizations and the calculatedstimulation indices improved from 1.8 to 2.8 against rVV-NS3 and from6.5 to 8.6 against rVV-Core with one vs. three immunizations. Nostimulation was observed against rVV-rastafar (encoding human Ras),confirming the antigen-specificity of the response induced by GI-5005.

FIG. 7 shows the results of chromium release assays performed withspleen effector cells derived from C57BL/6 and BALB/c mice that receivedone, two or three immunizations with GI-5005. Specifically, spleen cellsfrom C57BL/6 mice (FIGS. 7A and 7B) or BALB/c mice (FIGS. 7C and 7D)that received one, two or three weekly injections with 5 YU GI-5005 wereplaced in individual wells of 24-well flat-bottomed tissue cultureplates (10×10⁶/well) and stimulated in vitro with GI-5005 (1×10⁶/well)for 5 days. At the end of the IVS culture period, doubling dilutions ofthe spleen cell cultures were mixed with ten thousand 51Cr-labeled EL4lymphoma cells (FIGS. 7A and 7B) or P815 leukemia cells (FIGS. 7C and7D) that had been infected overnight with recombinant vaccinia virusencoding HCV NS3 (FIGS. 7A and 7C) or HCV Core (FIGS. 7B and 7D). E:Tratio refers to the effector:target cell ratio based on spleen effectorcell concentrations at the start of the IVS culture period. Results areexpressed as the average percent specific lysis +/−S.D. for triplicatesamples isolated after 6 hours of co-culture in 96-well, V-bottomedplates. Percent spontaneous ⁵¹Cr release was 10% for EL-4-rVV-N53, 10%for EL4-rVV-Core, 12% for P815-rVV-NS3 and 11% for P815-rVV-Core.Confirming the findings reported in FIG. 4, the results presented inFIG. 7 show dose-dependent killing on syngeneic tumor cell targetsinfected with either rVV-NS3 or rVV-Core which increases in proportionto the number of immunizations.

The results presented in FIG. 8 show the cytokine secretion profiles ofspleen cells derived from C57BL/6 and BALB/c mice that received one, twoor three immunizations with GI-5005 in response to in vitro stimulationwith GI-5005. Specifically, spleen cells from C57BL/6 mice (upperpanels) or BALB/c mice (lower panels) that received one, two or threeweekly injections with 5 YU GI-5005 were placed in individual wells of24-well flat-bottomed tissue culture plates (10×10⁶/well) and stimulatedin vitro with GI-5005 (1×10⁶/well). Cell-free supernatants werecollected at 48 hours after initiation of culture and cytokines werequantified by the University of Colorado Cancer Center Flow CytometerFacility using the flow-cytometer based Luminex™ assay (Biosource).IFN-g=IFN-γ; TNF-a=TNF-α. These results show that the cytokine responseof cells from immunized mice against yeast antigens is predominantly ofthe T_(H)1-like, pro-inflammatory variety and that more than oneimmunization is required to see the full spectrum of response. It isimportant to further note that the T_(H)2 cytokines IL-4 and IL-10 aregenerally not detected, suggesting that yeast vehicles of the inventionprimarily induce cellular rather than humoral immunity.

The data presented above indicated that immune responses induced byGI-5005 were enhanced by repeated weekly administrations. To exploreboosting of immune responses with GI-5005, the experiment outlined inTable 2 was undertaken. In brief, female BALB/c mice received fiveweekly injections of GI-5005 followed by no boosting or by boosting atweekly, bi-weekly, monthly or bimonthly intervals. Mice were sacrificed16 days after the last boosting. The results importantly show thatrepeated weekly immunization does not result in induction ofneutralization and/or tolerance in that even after 12 weekly injectionsa subsequent administration resulted in boosting as measured bylymphocyte proliferation and cell-mediated cytotoxicity assays.

TABLE 2 Immunization and boosting schedule with GI-5005 D D D D D D D DD D D D Group D 0 D 7 14 21 28 35 42 49 56 63 70 77 84 100 PBS control —— — — — — — — — — — — — Sacrifice No boost I I I I I — — — — — — — —Sacrifice 2 month boost I I I I I — — — — — — — I Sacrifice Monthlyboost I I I I I — — — I — — — I Sacrifice Bi-weekly boost I I I I I — I— I — I — I Sacrifice Weekly boost I I I I I I I I I I I I I Sacrifice I= immunization with GI-5005

FIG. 9 shows the results of a lymphocyte proliferation assay performedwith spleen cells derived from the BALB/c mice that received GI-5005 onthe immunization schedule outlined in Table 2. Briefly, spleen cellsfrom BALB/c mice that were immunized with 5 YU GI-5005 on the scheduleas described in Table 2 were placed in individual wells of 96-wellU-bottomed tissue culture plates (400,000 cells/well) and stimulated invitro with either nothing (Bkgd), GI-5005 (320,000 or 20,000 yeastcells/well), Concanavlin A (ConA; 2.5 μg/mL) orLipopolysaccharide+dextran sulfate (LPS+DS; 25 μg/ml and 20 μg/mL).3HTdR was added on day 3 and the plates were harvested 18 hoursthereafter. Results are expressed as the average CPM +/−S.D. fortriplicate samples. FIG. 9 shows that the boostable response againstyeast-associated antigens is quite evident and there is no apparentinduction of tolerance.

FIG. 10 shows the results of a chromium release assay performed withspleen effector cells derived from the BALB/c mice that were immunizedand boosted as described in Table 2. Briefly, spleen cells from BALB/cmice that were immunized with 5 YU GI-5005 on the schedule as describedin Table 2 were placed in individual wells of 24-well flat-bottomedtissue culture plates (10×10⁶/well) and stimulated in vitro with GI-5005(1×10⁶/well) for 5 days. At the end of the IVS culture period, doublingdilutions of the spleen cell cultures were mixed with ten thousand⁵¹Cr-labeled P815-N53 leukemia cells. E:T ratio refers to theeffector:target cell ratio. Results are expressed as the average percentspecific lysis +/−S.D. for triplicate samples isolated after 6 hours ofco-culture in 96-well, V-bottomed plates. Percent spontaneous 51Crrelease was 12% for P815-N53. Confirming the findings reported in FIG.9, the results presented in FIG. 10 show dose-dependent killing onsyngeneic tumor cells stably expressing HCV NS3 and further demonstratethe durability as well as boostability of the CTL response induced byGI-5005.

(e) Durability of Immune Responses Induced with GI-5005

In order to evaluate the robustness of the cellular immune responsesinduced upon immunization with GI-5005, C57BL/6 and BALB/c mice thatreceived three weekly doses of GI-5005 were sacrificed one month and twomonths post-dosing. FIG. 11 examines the durability of yeast-specificlymphocyte proliferation while FIG. 12 examines the durability of NS3-and Core-specific cytotoxic cell activity and FIG. 13 shows yeast—aswell as N53-specific cytokine secretion profiles. Overall, these resultssuggest that administration of GI-5005 induces memory T cell responsesthat are long lasting and robust.

Durability of Lymphocyte Proliferative Responses Induced with GI-5005

The results presented in FIG. 11 show that proliferative responsesagainst yeast antigens last at least 2 months following three weeklyimmunizations. Briefly, spleen cells from C57BL/6 (FIG. 11A) or BALB/c(FIG. 11B) mice that received either nothing (Nave) or three weeklyimmunizations with 5 YU GI-5005, and were rested for five (1 monthdurability) or nine (2 month durability) weeks prior to sacrifice, wereplaced in individual wells of 96-well U-bottomed tissue culture plates(400,000 cells/well) and stimulated in vitro with either nothing (Bkgd)or GI-5005, (400,000 yeast cells/well). ³HTdR was added on day 5 and theplates were harvested 18 hours thereafter. Results are expressed as theaverage CPM +/−S.D. for triplicate samples. It is important to notethese results examine yeast-specific as opposed to HCV NS3- orCore-specific proliferative responses as described in FIGS. 2 and 6. Thestimulation indices against yeast antigens in these particularexperiments range from approximately 11 to 77.

Durability of Cytotoxic Effector Cell Responses Induced with GI-5005

As shown in FIG. 12, and similar to results regarding lymphocyteproliferative responses, the durability of cytotoxic effector cellactivity induced with GI-5005 is at least two months. Briefly, spleencells from C57BL/6 (FIG. 12A) or BALB/c (FIG. 12B) mice that receivedeither nothing (Nave) or three weekly immunizations with 5 YU GI-5005,and were rested for five (1 month durability) or nine (2 monthdurability) weeks prior to sacrifice, were placed in individual wells of24-well flat-bottomed tissue culture plates (10×10⁶/well) and stimulatedin vitro with GI-5005 (1×10⁶/well) for 5 days. At the end of the IVSculture period, doubling dilutions of the spleen cell cultures weremixed with ten thousand ⁵¹Cr-labeled EL4-NS3 lymphoma cells (FIG. 12A)or P815-NS3 leukemia cells (FIG. 12B). E:T ratio refers to theeffector:target cell ratio based on spleen effector cell concentrationsat the start of the IVS culture period. Results are expressed as theaverage percent specific lysis +/−S.D. for triplicate samples isolatedafter 6 hours of co-culture in 96-well, V-bottomed plates. Percentspontaneous ⁵¹Cr release was 11% for EL-4-NS3 and 11% for P815-NS3.

Durability of Cytokine Secretion Responses Induced with GI-5005

FIG. 13 shows the durability of the cytokine secretion profiles ofspleen cells derived from C57BL/6 and BALB/c mice that received threeweekly immunizations with GI-5005 in response to in vitro stimulationwith GI-5005 and rVV-NS3. Briefly, spleen cells from C57BL/6 (FIGS. 13Aand 13B) or BALB/c (FIGS. 13C and 13D) mice that received nothing(Naïve) or three weekly immunizations with 5 YU GI-5005 and were restedfor five (1 month durability) or nine (2 month durability) weeks priorto sacrifice were placed in individual wells of 24-well flat-bottomedtissue culture plates (10×10⁶/well) and stimulated in vitro with GI-5005(1×10⁷/well) or rVV-NS3 (1×10⁷ pfu/well). Cell-free supernatants werecollected at 48 hours (IVS w/GI-5005) or 120 hours (IVS w/rVV-NS3) afterinitiation of culture. Cytokines were quantified using the Luminex™assay (Biosource). IFN-g=IFN-γ; TNF-a=TNF-α. These results show thatdurability of cytokine-secreting cells induced by immunization withGI-5005 is at least two months. In contrast to the yeast-specificprofile of cytokines, these data also show that the antigen-specific(i.e., NS3-specific) response, using rVV-NS3 as a stimulus, is limitedpredominantly to GM-CSF and IFN-γ.

(f) Comparison of Administration of Different Doses of GI-5005

The results summarized in FIGS. 14 and 15 compare the induction ofcytotoxic effector cells and cytokine-secreting cells respectively byGI-5005 Tarmogen™s that express different amounts of antigen. This studywas undertaken as part of the development of a potency assay.

In brief, GI-5005 Tarmogen™s were produced that express approximately1400, 500 and <50 ng/YU of HCV NS3-Core fusion protein. This wasaccomplished by varying the amount of copper present during theinduction period. The three Tarmogen™s are designated as GI-5005-H (1400ng/YU; 0.02 ng protein/ng total protein), GI-5005-M (500 ng/YU; 0.008 ngfusion protein/total protein) and GI-5005-L (<50 ng/YU; <0.001 ngprotein/ng total protein). Groups of five female BALB/c mice (H-2^(d))were immunized weekly with the three different GI-5005 Tarmogen™s atthree doses, 0.1, 1 and 10 YU. Mice were sacrificed seven days after thethird weekly injection and their spleen cells were subjected to in vitrostimulation (IVS) as will now be described.

In FIG. 14, spleen cells from the immunized mice, pooled by group, wereplaced into IVS separately with each of the three different GI-5005Tarmogen™s. Cell-mediated cytotoxic activity of the IVS cultures wasassessed on H-2^(d)-bearing P815 cells stably expressing HCV NS3.Briefly, spleen cells from BALB/c mice that received three weeklyinjections of 0.1, 1 or 10 YU of either GI-5005-H (FIGS. 14A-14C),GI-5005-M (FIGS. 14D-14F) or GI-5000-L (FIGS. 14G-14I) were placed inindividual wells of 24-well flat-bottomed tissue culture plates (10×10⁶spleen cells/well) and stimulated in vitro (IVS) with the indicatedGI-5005 Tarmogen™ (2×10⁶ yeast cells/well) for 5 days. At the end of theIVS culture period, doubling dilutions of the spleen cell cultures weremixed with ten thousand ⁵¹Cr-labeled P815-NS3 leukemia cells. E:T ratiorefers to the effector:target cell ratio based on spleen effector cellconcentrations at the start of the IVS culture period. Results areexpressed as the average percent specific lysis +/−S.D. for triplicatesamples isolated after 6 hours of co-culture in 96-well, V-bottomedplates. Percent spontaneous ⁵¹Cr release was 12% for P815-NS3. 10 YU, 1YU & 0.1 YU in the legend of each figure refer to the amount of GI-5005used for immunization. The data show clear dose responses based on asingle parameter; that is, the amount of HCV antigen being expressed inthe Tarmogen™™ used for immunization or for in vitro stimulation. Asimilar conclusion can be drawn from the data presented in FIG. 15.

FIG. 15 shows the levels of IL-6 secreted in response to yeast-specificantigens vs. GM-CSF secreted in response to HCV NS3-specific antigen.Specifically, spleen cells from BALB/c mice that received three weeklyinjections of 0.1, 1 or 10 YU (X-axis) of either GI-5005-H (FIG. 15A),GI-5005-M (FIG. 15B) or GI-5000-L (FIG. 15C) were placed in individualwells of 24-well flat-bottomed tissue culture plates (10×10⁶ spleencells/well) and stimulated in vitro (IVS) with either GI-5005-H (2×10⁶yeast cells/well) or rVV-NS3 (100×10⁶ pfu/well). Cell-free supernatantswere collected at 72 hours (IVS w/GI-5005) or 120 hours (IVS w/rVV-NS3)after initiation of culture. Cytokines were quantified using theLuminex™ assay (Biosource). In brief, these data indicate that theinduction of IL-6-secreting cells is dependent on the number ofTarmogen™s that are used for immunization but is independent of theamount of HCV antigen being expressed in the Tarmogen™™. In contrast,the induction of cells secreting GM-CSF is dependent on both criteria.Based on the data presented in FIGS. 14 and 15 a minimum of 500 ngfusion protein/YU or 0.008 ng protein/ng total protein is required forinducing an antigen-specific response.

Example 6

The following example shows non-clinical pharmacology studies in miceusing the GI-5005 Tarmogen™ expressing HCV antigens: tumor protectionand therapy studies.

Because an in vivo animal model of protection or therapy against HCV isnot available, the present inventors have used protection and therapyagainst HCV antigen-bearing tumors in vivo to demonstrate the activityof GI-5005.

(a) GI-5005 Induces Protective Immunity against NS3-Expressing TumorCells

The experiments described above demonstrate the immunogenicity ofGI-5005 in C57BL/6 and BALB/c mice. In order to determine if injectionof GI-5005 yeast elicited protective immunity, BALB/c mice were injectedsubcutaneously once a week for three weeks with 0.1, 0.7 or 5 YU ofeither GI-5005 or GI-5003 (a Tarmogen™ that expresses only HCV NS3protease), with 5 YU GI-4014 (a Tarmogen™ expressing a mutated Rasprotein) as a negative control, or with nothing. One week after thefinal immunization, the mice were challenged with subcutaneouslyinjected syngeneic A20 tumor cells stably transfected with HCV NS3(A20-NS3). Tumor volume was measured on day 21 after challenge. The datapresented in FIG. 16 show that the mice that were immunized with aTarmogen™ expressing HCV NS3 antigens, GI-5005 or GI-5003, wereprotected from challenge with A20-NS3 tumor cells, whereas miceimmunized with nothing or with GI-4014 were not. Results are expressedas the mean tumor volume +/−S.D. These results show that GI-5005 inducesdose- and antigen-dependent immune responses that protect mice fromsyngeneic tumor cells expressing HCV NS3.

This experiment was repeated in C57BL/6 mice that were injected weeklyfor three weeks with GI-5005 and challenged seven days thereafter withEL4-NS3 lymphoma cells injected subcutaneously. Briefly, C57BL/6 mice (5per group) were injected subcutaneously weekly for three weeks withnothing (Naive) or with 5 YU GI-5005. Mice were challenged 7 days afterthe final immunization with 5×10⁴ A20-NS3 injected subcutaneously.Tumors were measured on the indicated day after challenge. Results areexpressed as the mean tumor volume +/− S.D. Numbers refer to the numberof animals with measurable tumors (* Tumors excised from immunized micewere found to no longer express NS3). The results presented in FIG. 17show that mice injected with GI-5005 were protected from challenge withEL4-NS3 whereas naïve mice were not. Injection of GI-5005 did notprotect mice from challenge with EL4 alone indicating that protectiveimmunity was antigen-specific (data not shown). To determine whether thetumors that had grown in the immunized mice were still expressing HCVNS3, the tumors were excised from the two GI-5005 immunized mice thatshowed evidence of tumor growth, as well as from the five naïve mousecontrols, and placed in tissue culture medium containing the antibioticG4.18. In EL4-NS3, the mammalian expression vector encoding HCV NS3 alsocontains a neomycin resistance gene that allows transfectants to grow inthe presence of the neomycin analog G4.18, thereby maintaining stableexpression of HCV NS3. Whereas EL4-NS3 tumor cells excised from naïvemice grew out in the presence of G4.18, tumors from theGI-5005-immunized mice did not. This observation suggests that there wasimmunological pressure to eliminate expression of the transfectedantigen. These observations indicate that GI-5005 induces protectiveimmune responses in vivo against challenge with syngeneic tumor cellsexpressing NS3.

(b) Immune Responses in “Protected” Mice

The availability of “protected” mice that had rejected syngeneic tumorcells expressing HCV NS3 provided the opportunity to examineantigen-specific immune responses in the setting of protective immunity.Spleen cells from the five mice described above that rejected EL4-NS3tumor cells were pooled and placed in individual wells of 96-wellU-bottomed tissue culture plates (4×10⁵ cells/well) and stimulated witheither nothing, GI-1001 (2×10⁵ yeast cells/well), GI-5005 (2×10⁵ yeastcells/well), rVV-Gag (1×10⁵ pfu/well), rVV-NS3 (1×10⁵ pfu/well), orrVV-Core (1×10⁵ pfu/well). ³HTdR was added on day 5 and the cells wereharvested 18 hours thereafter. Results are expressed as the average CPM+/−S.D. for quadruplicate samples. FIG. 18 shows the proliferativeresponse of spleen cells derived from protected mice to yeast-specific,as well as HCV NS3- and HCV Core-specific stimuli. FIG. 19 examinestheir cytotoxic effector cell activity. In this experiment, spleen cellsfrom the five immunized mice that rejected EL4-NS3 tumor cells or fromnaïve mice were pooled together and placed in individual wells of24-well flat-bottomed tissue culture plates (8×10⁶ spleen cells/well)and stimulated in vitro (IVS) with either GI-5005 (1×10⁶ yeastcells/well) or rVV-NS3 (8×10⁵ pfu/well) for 5 days. At the end of theIVS culture period, doubling dilutions of the spleen cell cultures weremixed with ten thousand ⁵¹Cr-labeled EL4 target cells that had beeninfected overnight with rVV-NS3. E:T ratio refers to the effector:targetcell ratio based on spleen effector cell concentrations at the start ofthe IVS culture period. Results are expressed as the average percentspecific lysis +/−S.D. for triplicate samples isolated after 5 hours ofco-culture in 96-well, V-bottomed plates. Percent spontaneous ⁵¹Crrelease was 33% for EL4-rVV-NS3. Taken together, these findings suggestthat protected mice, i.e. immunized mice that rejected NS3-expressingtumor cells, have enhanced immune responses to HCV NS3 as compared tomice that were simply immunized as shown in Example 5 above.

(c) GI-5005 Stimulates Cytotoxic Effector Cell Activity in Spleen CellsIsolated from Naïve Tumor-Bearing Mice

The results presented above suggest that exposure of GI-5005 mice to asecondary source of HCV antigen, namely tumor cells expressing HCV NS3,results in a boosting effect as evidenced by enhanced proliferative andcytotoxic effector cell responses. In order to determine if GI-5005yeast could further stimulate T cell activity from antigen-bearing mice,thus mimicking T cell activation in chronic HCV-infected patients, naïveC57BL/6 mice were injected subcutaneously with EL4-NS3 tumor cells.After 3 weeks, when tumor volumes reached approximately 2500 mm³, themice were sacrificed and spleen cells were incubated with either vectorcontrol (GI-1001) or GI-5005 yeast. Cytotoxic effector cell activityagainst rVV-NS3 infected EL4 target cells was assessed six days afterinitiation of in vitro stimulation. Specifically, spleen cells from fivenaive mice that were injected with EL4-NS3 tumor cells 21 dayspreviously were pooled together and placed in individual wells of24-well flat-bottomed tissue culture plates (8×10⁶ spleen cells/well)and stimulated in vitro (IVS) with either GI-1001 or GI-5005 (1×10⁶yeast cells/well) for 6 days. At the end of the IVS culture period,doubling dilutions of the spleen cell cultures were mixed with tenthousand ⁵¹Cr-labeled EL4 target cells that had been infected overnightwith rVV-NS3. E:T ratio refers to the effector:target cell ratio basedon spleen effector cell concentrations at the start of the IVS cultureperiod. Results are expressed as the average percent specific lysis+/−S.D. for triplicate samples isolated after 5 hours of co-culture in96-well, V-bottomed plates. Percent spontaneous ⁵¹Cr release was 33% forEL4-rVV-NS3. The results presented in FIG. 20 show that GI-5005 canstimulate cytotoxic effector cells derived from mice bearing tumorsexpressing HCV-NS3.

(d) GI-5005 Induces Therapeutic Activity against NS3-Expressing TumorCells

The results presented in FIG. 20 show that GI-5005 can re-stimulateNS3-specific cytotoxic effector activity from spleen cells of C57BL/6mice bearing EL4-NS3-expressing tumors. This suggests that a therapeuticeffect might also be attainable. To assess this possibility, BALB/c mice(5 per group) were injected subcutaneously with syngeneic 1.25×10⁵A20-NS3 B lymphoma cells stably transfected with DNA encoding HCV NS3.Beginning seven days after tumor implantation, the mice were immunizedonce a week for three weeks with either PBS or with YU GI-5005. Tumorgrowth was monitored and the mice were sacrificed 28 days after tumorimplantation when the tumors in the PBS group reached 2500 mm³. Resultsin FIG. 21 are expressed as the mean tumor volume +/−S.D and numbersrefer to the number of animals with measurable tumors (* Tumors excisedfrom all tumor bearing mice were found to still express NS3).

FIG. 21 shows that therapeutic administration of GI-5005 results intumor remission. In brief, whereas all five tumor-bearing mice that weretreated with PBS showed tumor growth, only three out five that weretreated with GI-5005 exhibited tumor growth and the tumors that arose inthe treated animals appeared to be growing much more slowly (mean tumorvolume in tumor-bearing mice in the PBS treated group was 2488+/−636 vs.1264+/−548 mm³ in the GI-5005 treated group). However, in contrast tothe results obtained with EL4-NS3 as described above, the HCV NS3protein was still being expressed in all of the tumors from A20-NS3tumor bearing mice (data not shown).

The immunotherapeutic property of GI-5005 was confirmed in a secondstudy as shown in FIG. 22 in which the number of implanted tumor cellswas varied. Briefly, BALB/c mice (5 per group) were injectedsubcutaneously with 2.5×10⁴, 5.0×10⁴, or 1×10⁵ A20-NS3 B lymphoma cells.Mice were therapeutically immunized by subcutaneous injection at skinsites distal to the tumor on days 7, 14 and 21 after tumor implantationwith either PBS or with 10 YU GI-5005. Tumor volume was measured on theindicated day after initiation of therapy. Results are expressed as themean tumor volume +/−S.D (FIG. 22A) and as the percentage of tumorbearing mice (FIG. 22B) on day 24 after initiation of therapy.

Example 7

The following example describes toxicity studies with the yeast vaccinesof the present invention.

As described above, the GI-5005 Tarmogen™ has been administered to morethan 300 mice in a number of different studies to date and no grosslyobservable toxicity has been evident. Other related products using theyeast-based vaccine platform have been administered to mice, rats,rabbits, pig-tailed and rhesus macaque monkeys with no major observabletoxicity. Because of the similarity, and therefore relevance of safetydata, of other yeast based products to the GI-5005 Tarmogen™, a numberof non-clinical safety assessments with these other Tarmogen™s aredetailed following the toxicity data or GI-5005.

The objective of this study was to determine the toxic effects ofGI-5005 in male and female New Zealand rabbits following once weeklysubcutaneous administration at a fixed dose volume of 1 mL for up tothirteen consecutive weeks (dosing on Days 1, 8, 15, 22, 29, 36, 43, 50,57, 64, 71, 78, 85 and 92), followed by specified recovery/necropsyintervals (Table 3). The dose levels were selected on the basis ofavailable data from previous studies. The subcutaneous route is theintended route of administration of this test article in humans. Theinterim report as summarized below. Three treatment groups (Groups 2 to4) of five male and five female New Zealand White rabbits wereadministered the test article at respective dose levels of 1, 10 and 100Yeast Units (YU). A control group (Group 1) of five animals/sex receivedthe vehicle, sterile phosphate buffered saline (PBS). The test articleor vehicle was administered once on Days 1, 8, 15, 22, and 29.Additionally, three treatment groups (Groups 6 to 8) of fiveanimals/sex/group (low and middle dose groups) and ten animals/sex/group(high dose group) were administered the test article at respective doselevels of 1, 10 and 100 YU. A control group (Group 5) of ten animals/sexreceived the vehicle PBS. In groups 5-8, the test article or vehicle wasadministered once on Days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78,85, an 92. Five animals/sex of groups 5-8 were sacrificed on Day-94. Theremaining 5 animals/sex in Groups 5 and 8 were maintained for a recoveryperiod of approximately 23 days.

TABLE 3 Rabbit GLP toxicity study design DOSE TERMINAL TERMINAL RECOVERYLEVEL NECROPSY NECROPSY NECROPSY Yeast Units INITIAL (DAY 31) (Day 97)(DAY 120)* GROUP NUMBER TREATMENT (YU)* M/F M/F M/F M/F 1 PBS 0 5/5 5/52 GI-5005 1 5/5 5/5 3 GI-5005 10 5/5 5/5 4 GI-5005 100 5/5 5/5 5 PBS 010/10 5/5 5/5 6 GI-5005 1 5/5 5/5 7 GI-5005 10 5/5 5/5 8 GI-5005 10010/10 5/5 5/5 *The test and control articles will be administered assingle subcutaneous injections (1.0 mL total volume) on Days 1, 8, 15,22, 29, 36, 43, 50, 57, 64, 71, 78, 85, and 92. One yeast unit equals 10million heat-killed yeast cells. For reporting purposes, yeast unitswill be abbreviated as YU.

All animals were observed for morbidity, mortality, injury, andavailability of food and water twice daily. Detailed clinicalexaminations, injection site irritation evaluations, opthalmoscopicexaminations, and body weight and food consumption measurements wereconducted during the course of the study. Clinical pathology evaluations(hematology, clinical chemistry, and urinalysis) were conducted on allsurviving animals predose, the day following each dose, and for animalsin Groups 1 to 4 at the Day 31 necropsy. Additional blood samples werecollected from all surviving animals predose and 1 hour postdose forserum antibody analysis and for animals in Groups 1 to 4 at the Day 31necropsy for serum antibody analysis. At the Day 31, Day 97 and Day 120necropsies, all animals in the appropriate groups were euthanized andcomplete macroscopic and microscopic examinations were conducted, alongwith protocol-designated organ weight measurements.

No treatment-related effects on survival, clinical findings, foodconsumption, opthalmology, or organ weights were observed.Microscopically, treatment-related changes were observed at theinjection sites of both sexes at all dose levels, and included fibrosis,subacute inflammation, and necrosis. In addition, findings ofgranulomatous inflammation were also noted in some but not all of theinjection sites. The incidence and severity of these findings weregenerally dose related. Granulocytic hyperplasia in the bone marrow offemales at 1 YU and both sexes at 10 and 100 YU, and follicular lymphoidhyperplasia and/or reactive red pulp/stromal hyperplasia in the spleenof females at 10 YU and both sexes at 100 YU were considered a secondaryresponse to the observed inflammation at the injection sites. Thesefindings correlated with the microscopic findings of tissue thickeningat the injection sites. Treatment related irritation, consisting of botherythema and edema, was observed at the injection sites of both sexes at100 YU, Minimal findings were also noted in both sexes at 10 YU,suggesting a relationship to treatment. There was no indication of anysign of recovery following dosing at any of the injection sites.Although the effect was a modest, a loss of body weight was noted inboth sexes at 100 YU, suggesting a relationship to treatment withGI-5005.

Treatment-related effects in hematology and clinical chemistry wereobserved and were considered secondary to the local inflammatoryresponses observed at the injection sites. Treatment-related increasesin leukocyte counts, reflecting increases in neutrophil counts, werenoted in all GI-5005-treated groups, with the onset and severitygenerally dose related. Some recovery in neutrophil levels was notedprior to the next dose. Treatment-related increases in globulin valueswere observed, with the onset and severity generally dose related. Theincreases tended to be progressive over time, with no indication ofrecovery.

Based on the conditions and findings of this study, administration ofGI-5005 at dose levels of 1, 10 and 100 YU to male and female rabbitsdid not result in any apparent systemic toxicity. Primary treatmentrelated findings were limited to local effects of fibrosis, subacuteinflammation, and necrosis at the injection sites, which were infrequentand mild to moderate except at the highest dose tested. Injection sitereactions may represent a potential dose limiting effect in the clinicalsetting. Concomitant increases in neutrophil counts and globulin valuesthat were considered secondary to the local inflammatory response.

To demonstrate the immunogenicity of GI-5005 in the rabbit toxicitystudy, and therefore immuno-toxicologic relevance of the study, alymphocyte proliferation assay was performed. While there are nostandardized methods for assaying lymphocyte proliferation in rabbits, anon-optimized assay of lymphocyte proliferation in response to yeastproteins was performed using lymphocytes isolated from ileocecocolic andaxillary lymph nodes harvested two days after the fifth immunization(Day 31). Lymph node cell suspensions from individual rabbits wereplaced in tissue culture in 96-well U-bottomed plates (4×10⁵ per well)with the indicated number of heat-killed GI-5005 yeast cells. Lymphocyteproliferation was determined on day 3 of culture by pulsing with 1μCi/well of ³H-TdR for 18 hr. Average stimulation indices obtained withmale (FIG. 23A) vs. female (FIG. 23B) lymph node cells are shown in FIG.23 (Results are presented as average stimulation indices +/−S.E.M.obtained for evaluable lymph node cell samples from individual rabbitswithin each dose group). Overall, the data show that only 1 out of 10rabbits immunized with the vehicle, PBS, showed a stimulation index ofgreater than 10 against the GI-5005 yeast, whereas 9 out of 10, 7 out of10 and 8 out of 10 the rabbits immunized with 1, 10 or 100 YU GI-5005respectively responded with a stimulation index of greater than 10. Nodifferences between the response of male versus female rabbit lymph nodecells could be discerned and a dose-response effect was not apparent.

An enzyme linked immunosorbent assay (ELISA) was used to detect andtiter anti-Saccharomyces cerevisiae antibodies (ASCA) in the sera ofrabbits (groups 1-4) that were injected with GI-5005 as part of MPIstudy 962-003. The serum samples examined were obtained on day 1, priorto the first injection, and on day 29, prior to the fifth weeklyinjection. All rabbits displayed ASCA titers of less than 1:100 at theinitiation of the study (Table 4). In contrast, all rabbits thatreceived GI-5005 showed elevated ASCA titers after administration offour weekly injections. However, the titers were low, less than1:10,000, and no dose-response effect was observed.

TABLE 4 Summary of anti-Saccharomyces cerevisiae antibody (ASCA) unitsin sera of rabbits (Groups 1-4) that were injected with GI-5005 as partof MPI Study 962-003 Mean ASCA Mean ASCA Mean ASCA units Injection units(day 1) units (day 15) (day 29) PBS 4 +/− 5 3 +/− 4 6 +/− 8 GI-5005 (1YU) 4 +/− 7 3 +/− 4 84 +/− 43 GI-5005 (10 YU) 2 +/− 2  6 +/− 10 127 +/−162 GI-5005 (100 YU)  6 +/− 10 19 +/− 17 165 +/− 84  Positive rabbitantiserum* 311 +/− 85  311 +/− 85  311 +/− 85  *A 1:1000 dilution of thepositive control rabbit antiserum contained 311 +/− 85 ASCA units whenrun in this assay suggesting that within 95% confidence an observed ASCAunit value of less than 300 would represent a titer of less than 1:1000.Averaged data +/− S.D. is shown in the following table.

The presence of HCV-NS3- and Core-specific serum antibodies produced inrabbits immunized weekly for five weeks with PBS or with 1, 10 or 100 YUof the GI-5005 Tarmogen™ were qualitatively evaluated by Western blotanalysis to gain a better understanding of the humoral antibodyresponses induced against the heterologous protein contained in thisTarmogen™. No HCV-specific antibodies were observed in sera obtainedfrom any animals prior to immunization. In contrast, antibodies reactingspecifically with NS3 and Core proteins were detected in serum samplesfrom 7 of 9 tested rabbits at Day 31 after receiving 5 weekly doses of100 YU GI-5005, and in serum samples from 1 of 3 animals in the YU dosegroup. No HCV-specific antibodies were detected in serum samples fromthe PBS or 1 YU groups at day 31. This analysis shows that adose-dependent induction of serum antibodies directed against theheterologous HCV NS3-Core protein contained in GI-5005 occurs as aresult of subcutaneous administration of this Tarmogen™ in rabbits.

The preliminary 97 day and 120 day clinical pathology and grossobservation data are consistent with the findings from the 31 daycohort. Thirteen weekly administrations of GI-5005 at dose levels of 1,10 and 100 YU to male and female rabbits did not result in any apparentsystemic toxicity. Primary treatment related findings were limited tolocal site reactions with the incidence and severity generally doserelated. However, in the 100 YU dose group more severe granulomatouschanges, fibrosis, and necrosis were observed in the injection sitereactions, and may represent a potential dose limiting effect in theclinical setting. Histopathological analysis of this 97 day cohort isnot yet available. Treatment-related increases in leukocyte counts,reflecting increases in neutrophil counts, and increases in globulinvalues were also observed, with the onset and severity generally doserelated for both effects. The increases tended to be progressive overtime, with no indication of recovery.

Gross safety assessments from 331 C57BL/6 and BALB/c mice injected withGI-5000 Tarmogen™ series products and prototypes showed notreatment-related deaths and mild to moderate hair loss and inflammationwith occasional ulceration consistent with delayed-type hypersensitivityat the site of injection in approximately 5% of animals. Injection sitereactivity was limited to C57BL/6 mice that are typically more sensitiveto skin trauma and may have been secondary to grooming behaviorsresulting from group housing conditions. No other gross clinicalabnormalities or adverse reactions were observed.

TABLE 5 Summary of safety studies performed with GI-5005 Tarmogen ™s andprototypes Species Study type tested Conclusions Safety assessments MiceMore than 300 mice have been injected with heat-inactivated intact yeastvia the subcutaneous route. No adverse effects have been observed at theinjection site, with the exception of mild to moderate skin reactivitynoted in approximately 5% of C57BL/6 mice, and no harmful effects havebeen observed at any time in any mice at doses as high as 10 YU. 28-dayGLP safety Rabbits Weekly administration of a total of five doses ofGI-5005 at study dose levels of 1, 10, and 100 YU to male and femalerabbits did not result in any apparent systemic toxicity. Adversereactions were limited to mild to moderate injection site reactions. Thestudy animals tolerated the treatment regimen well. 97-day GLP safetyRabbits The preliminary 97 day clinical pathology and gross studyobservation data are consistent with the findings from the 31 daycohort. No apparent systemic toxicity. Primary treatment relatedfindings were limited to local site reactions with the incidence andseverity generally dose related. Histopathological analysis of this 97day cohort is not yet available. Treatment-related increases inleukocyte counts, reflecting increases in neutrophil counts, andincreases in globulin values were also observed, with the onset andseverity generally dose related for both effects.

Each publication described or cited herein is incorporated herein byreference in its entirety.

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While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. A composition comprising: a) a yeast vehicle; and b) an HCV fusionprotein comprising HCV sequences, wherein the HCV sequences consist ofbetween one and five HCV proteins or immunogenic domains thereof,wherein the HCV proteins are selected from the group consisting of: HCVCore (positions 1 to 191 of SEQ ID NO:20); HCV E1 envelope glycoprotein(positions 192 to 383 of SEQ ID NO:20); HCV E2 envelope glycoprotein(positions 384 to 746 of SEQ ID NO:20); HCV P7 ion channel (positions747 to 809 of SEQ ID NO:20); HCV NS2 metalloprotease (positions 810 to1026 of SEQ ID NO:20); HCV NS3 protease/helicase (positions 1027 to 1657of SEQ ID NO:20); HCV NS4a NS3 protease cofactor (positions 1658 to 1711of SEQ ID NO:20); HCV NS4b (positions 1712 to 1972 of SEQ ID NO:20); HCVNS5a (positions 1973 to 2420 of SEQ ID NO:20); and HCV NS5bRNA-dependent RNA polymerase (positions 2421 to 3011 of SEQ ID NO:20);wherein the composition elicits an immune response against each of theHCV proteins or immunogenic domains thereof in the HCV fusion protein.2. The composition of claim 1, wherein the fusion protein is expressedby the yeast vehicle.
 3. The composition of claim 1, wherein the HCVsequences consist of an HCV NS3 protease sequence or a portion thereofcomprising at least one immunogenic domain linked to an a C-terminaltruncated HCV Core sequence or a portion thereof comprising at least oneimmunogenic domain, wherein the HCV NS3 protease sequence lacks thecatalytic domain of a natural HCV NS3 protease.
 4. The composition ofclaim 1, wherein the HCV fusion protein comprises an HCV NS3 proteasesequence linked to a heterologous amino acid sequence, wherein the HCVNS3 protease sequence consists of positions 1027 to 1657 of SEQ IDNO:20, wherein the serine residue corresponding to position 1165 of SEQID NO:20 has been mutated to inactivate the proteolytic activity of theprotein.
 5. The composition of claim 1, wherein the HCV sequencesconsist of a transmembrane domain-deleted HCV NS4b protein or at leastone immunogenic domain thereof.
 6. The composition of claim 1, whereinthe HCV sequences consist of: an HCV NS3 protein or at least oneimmunogenic domain thereof fused to an HCV Core protein or at least oneimmunogenic domain thereof fused to an NS5b protein or at least oneimmunogenic domain thereof.
 7. The composition of claim 1, wherein theHCV sequences consist of inactivated HCV NS3 or at least one immunogenicdomain thereof fused to HCV NS4a or at least one immunogenic domainthereof fused to HCV NS4b lacking a transmembrane domain or at least oneimmunogenic domain thereof.
 8. The composition of claim 1, wherein theHCV sequences consist of an HCV NS5a protein or at least one immunogenicdomain thereof fused to an HCV NS5b protein containing an inactivatingdeletion of NS5b C-terminus or at least one immunogenic domain thereof.9. The composition of claim 1, wherein the HCV fusion protein consistsof an amino acid sequence selected from the group consisting of: SEQ IDNO:4, SEQ ID NO:8, SEQ ID NO:16 and SEQ ID NO:18.
 10. The composition ofclaim 1, further comprising at least one biological response modifier.11. The composition of claim 1, wherein the yeast vehicle is a wholeyeast.
 12. The composition of claim 11, wherein the whole yeast iskilled.
 13. The composition of claim 11, wherein the whole yeast isheat-inactivated.
 14. The composition of claim 11, wherein the wholeyeast is from Saccharomyces cerevisiae.
 15. A method to reduce hepatitisC virus (HCV) infection or at least one symptom resulting from HCVinfection in a subject, comprising administering to a subject that hasbeen infected with HCV or is at risk of being infected with HCV, acomposition according to claim 1, wherein administration of thecomposition to the subject reduces HCV infection or at least one symptomresulting from HCV infection in a subject.
 16. The method of claim 15,wherein administration of the composition to the subject reduces HCVviral load in the subject.
 17. The method of claim 15, whereinadministration of the composition to the subject reduces liver damage inthe subject.
 18. The method of claim 15, further comprisingadministering to the subject at least one biological response modifier.19. The method of claim 15, further comprising administering to thesubject one or more additional compounds useful treating or amelioratinga symptom of HCV infection.
 20. A method to elicit an antigen-specific,cell-mediated immune response against an HCV antigen, comprisingadministering to a subject a composition according to claim 1.